HUP0203974A2 - Improved dna vaccines for production-type animals - Google Patents

Abstract

The subject of the invention is a DNA vaccine against a pathogen that infects domestic animals, especially cattle and pigs, which contains a plasmid containing a nucleotide sequence encoding an immunogen of a pathogen of the given animal species in a way that enables the sequence to be expressed in vivo, and also a cationic quaternary ammonium salt of the general formula (I) contains a lipid, where R1 in the formula is a saturated or unsaturated straight-chain aliphatic group with 12-18 carbon atoms, R2 is another aliphatic group with 2-3 carbon atoms, and X is a hydroxyl or amino group, and this lipid is preferably DMRIE. HE

Description

P02 03974

Representative:

Danube

PUBLICATION COPY

Patent and Trademark Office Ltd.

Budapest

Improved DNA vaccines for pets

The invention relates to improved DNA vaccines for domestic animals, particularly bovines and porcines.

The use of deoxyribonucleic acid (DNA) molecules as vaccines has been known since the early 1990s [Wolf et al., Science 247, 1465-1468 (1990)]. In such vaccination procedures, the cells of the subject to be vaccinated are transfected in vivo with DNA or RNA molecules encoding immunologically active proteins, which induces cellular and humoral immunity.

DNA vaccines comprise at least one plasmid which can be expressed by a mechanism in the cells of the subject to be vaccinated, and a pharmaceutically acceptable carrier or excipient. The nucleotide sequence of the plasmid encodes, inter alia, one or more immunogens, i.e. proteins or glycoproteins which induce a cellular immune response (mobilization of T lymphocytes) and a humoral immune response (specific response to the immunogen) in the subject to be vaccinated.

Our file number: 97287-2709/SG) are able to induce the production of specific antibodies [Davis, H.L., Current Opinion Biotech. 8^, 635-640 (1997)].

Not all immunogens derived from pathogens behave as antigens that are sufficiently effective on their own to induce an optimal protective immune response in the animal to be vaccinated. Therefore, it is necessary to enhance the immune response.

Several routes of administration have been proposed for DNA vaccines (intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, transmucosal, and the like). Various delivery devices have also been proposed, in particular DNA-coated gold particles that are fired to enter the skin cells of the subject to be vaccinated [Tang et al., Nature 356, 152-154 (1992)], and liquid jet injectors that can simultaneously transfect cells in the skin and subcutaneous tissues [Furth et al., Analytical Bioch. 205, 365-368 (1992)].

The following compounds were used for in vitro DNA transfection:

A) - cationic lipids

Cationic lipids can be divided into the following four subgroups:

1) Cationic lipids containing quaternary ammonium salts, such as DOTMA (dioleoyloxypropyltrimethylammonium; manufactured by Gibco, trade name: Lipofectine), DOTAP [trimethyl2,3-(octadec-9-enoyloxy)-1-propaneammonium; Gregoriadis et al., FEBS Letters 402, 107-110 (1997)], DMRIE [N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propaneammonium; WO-A-9634109], DLRIE [N-(2-hydroxyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaneammonium; Feigner et al., Ann. N.Y. Acad. Sci. 772, 126-139 (1995)].

Cationic lipids containing quaternary ammonium salts can be combined with neutral lipids such as DOPC (dioleoylphosphatidylcholine) or DOPE (dioleoylphosphatidylethanolamine) [J.P. Behr, Bioconjugate Chemistry 5, 382-389 (1994)].

2) Lipoamines, such as DOGS [dioctadecylamidoglycylspermine; manufacturer: Promega, trade name: Transfectam; Abdallah et al., Biol. Cell. 85, 1-7 (1995)], DC-Chol [dimethylaminoethanecarbamoylcholesterol; Gao and Hunag, Biochem. Biophys. Res. Commun. 179, 280-285 (1991)], BGSC (bis-guanidine-tren-cholesterol) [Vigneron et al., Proc. Natl. Acad. Sci. USA 93, 9682-9686 (1996)].

3) Cationic lipids containing quaternary ammonium salts and lipoamines, such as DOPSA [N,N-dimethyl-N-(2(sperminecarboxamido)ethyl)-2,3-bis(dioleoyloxy)-1-propanimide pentahydrochloride; sold by Gibco under the trade name LipotectAmine®; Hawley-Nelson et al., Focus 15, 73-79 (1993)], GAP-DLRIE [N-(3-aminopropyl)-N,N-dimethyl-2,3bis(dodecyloxy)-1-propanammonium; Wheeles et al., Proc. Natl. Acad. Sci. USA 93, 11454-11459 (1996); Norman et al., Vaccine 15, 801-803 (1997)].

4) Lipids containing amidine salts, such as ADPDE and ADODÉ [Ruysschaert et al., Biochem. Biophys. Res Commun. 203, 1622-1628 (1994)].

B) - polymers, such as SuperFect® [molecules of activated dendrimers; manufactured by Qiagen; Xu et al., Mol. Genet. Metab. 64, 193-197 (1998)], and

C) - biochemical agents, such as toxins, especially cholera toxin.

Some of the above compounds have been used with more than moderate success in DNA vaccine formulations. The knowledge gained in the field of in vitro transfection is not transferable to vaccination with DNA vaccines, where the ultimate goal is to provide a protective immune response. Negative effects on the induction of effective immune protection have also been observed with the use of compounds known to promote transfection in vitro. Some formulating compounds are toxic to transfected cells at high doses.

In the work of Etchart [Etchart et al., J. Gen. Virol. 78, 157-1580 (1997)], DOTAP had no adjuvant effect when DNA vaccines were administered intranasally, but did have an adjuvant effect when administered orally. In intranasally administered mouse models, DOTAP was also used in DNA vaccines encoding influenza virus hemagglutinin (HA) [Ban et al., Vaccine 15, 811-813 (1997)], but the addition of DOTAP inhibited the immune response. Intramuscular administration of DC-Chol or DOTAP/DOPE in mouse models treated with DNA vaccines encoding the hepatitis B virus surface protein (S) increased the antibody response, whereas Lipofectin (or DOTMA) did not [Gregoriadis et al., FEBS Letters 402, 107-110 (1997)]. Intramuscular administration of DCChol/DOPE in mouse models treated with DNA vaccines against human immunodeficiency virus (HIV, Env protein) induced a more effective immune response, but did not increase it when administered subcutaneously or intradermally [Ishii et al., AIDS Res. Hum. Retro. 13, 1421-1428 (1997)].

The immune response induced by DNA vaccines in particular can also be enhanced by the administration of certain cytokines, especially interleukins or interferons. Each cytokine induces a specific reaction characteristic of it and shifts the immune response to a greater or lesser extent towards cellular or humoral responses [Pasquini et al., Immunol. Cell. Biol. 75, 397-401 (1997); Kim et al., J. Interferon Cytokine Res. 19, 77-84 (1999)]. The adjuvant effect of a cytokine produced from a given species is not necessarily the same in different immunological environments, especially when the cytokine is administered to another species, i.e. in a heterologous immune system. It may happen that the administered cytokine has no adjuvant effect and may even have the opposite effect to that desired, i.e. it may reduce or inhibit the immune response. A DNA vaccine encoding a single-chain immunoglobulin fused to GM-CSF does not therefore enhance the immune response, whereas if the fusion protein is administered directly to mice, it will be as effective as if a fusion protein consisting of Fv and the cytokine IL-1beta or the DNA vaccine encoding it had been administered.

[Hakim et al., J. Immunol. 157, 5503-5511 (1996)]. The use of plasmids coexpressing the cytokine IL-2 and the hepatitis B virus envelope protein in fused or unfused form enhances both humoral and cellular immune responses [Chow et al., J. Virol. 71, 169-178 (1997)]. However, bicistronic plasmids encoding the human immunodeficiency virus (HIV-1) gpl20 glycoprotein and the cytokine IL-2 induced a lower specific anti-gpl20 immune response than monocistronic plasmids encoding gpl20 alone [Barouch et al., J. Immunol. 161, 1875-1882 (1998)]. Coinjection of two expression vectors into mice, one encoding the G glycoprotein of rabies virus and the other encoding mouse GMCSF, stimulates the activity of both B and T lymphocytes, whereas coinjection of a plasmid encoding gamma interferon instead of mouse GM-CSF results in a decreased immune response [Xiang et al., Immunity 2' 129-135 (1995)].

Certain modifications of antigens, such as deletion of parts of the nucleotide sequence encoding the antigen, or insertion of a DNA fragment into the nucleotide sequence encoding the antigen or into the untranslated regions located 3' or 5' therefrom, may also increase the efficacy of DNA vaccines, in particular by enhancing antigen expression or presentation.

In practice, however, manipulation of the nucleotide sequence encoding the antigen may result in a reduction or loss of the original immunological activity. Thus, when the transmembrane region of the gene encoding the G antigen of rabies virus was deleted and a DNA vaccine encoding this modified antigen was administered intramuscularly, the immune protection induced in a mouse model was reduced [Xiang et al., Virol. 209, 569 (1995)]. Deletion of the transmembrane region of the gene encoding the gD glycoprotein of bovine herpesvirus (BHV) did not allow an increase in the antigenic response and induced only partial protection in cattle vaccinated intramuscularly [van Drunen-Little-van den Hurk et al., J. Gen. Virol. 79, 831-839 (1998)]. In guinea pigs, it provided identical humoral and cellular immune responses and immune protection when the animals were immunized with a DNA vaccine encoding the GP glycoprotein of Ebola virus or with a DNA vaccine encoding the secreted form of the same glycoprotein.

Insertion of the human tissue plasminogen activator (tPA) signal sequence into the malaria Pf332 antigen failed to enhance the antigenic response in mice vaccinated intramuscularly [Haddad et al., FEMS 18, 193202 (1997)]. Addition of the tPA sequence in a phase-locked manner to the murine rotavirus VP7 antigen and intradermally inoculating mice with it also failed to enhance the antigenic response, while a fusion protein formed from the VP4 antigen and tPA did enhance the antigenic response but did not induce effective protection [Choi et al., Virology 250, 230-240 (1998)].

Modifications made to the nucleotide sequence of a given antigen cannot generally be directly extrapolated to another antigen, as antigens do not always have the same structural makeup.

The aim of the invention is to increase the effectiveness of DNA vaccines. More specifically, to generate a better immune response and particularly effective protection in domestic animals, especially cattle and pigs, using DNA vaccines.

It is a further object of the invention to provide improved DNA vaccines that induce an effective and protective immune response in cattle against bovine herpesvirus type 1 (BHV-1), also known as infectious bovine rhinotracheitis (IBR), bovine respiratory syncytial virus (BRSV), mucosal disease virus or bovine pestivirus type 1 or 2 (bovine viral diarrhea virus or BVDV-1 or BVDV-2) and parainfluenza virus type 3 (bPI-3).

The invention also aims to produce improved DNA vaccines that induce an effective and protective immune response in pigs, which contain at least one valence of the following: porcine herpesvirus or Aujeszky's disease (pseudorabies virus or PRV), porcine reproductive respiratory syndrome virus (or PRRSV), swine influenza virus (or SIV), traditional hog cholera virus (or HCV), parvoviruses.

The invention also aims to produce improved DNA vaccines that allow the development of effective and protective immunity in cattle and that contain at least one valency of the following: BHV-1, BRSV, BVDV, bPI-3 and rabies viruses.

The invention relates to an improved DNA vaccine which enables effective protection against at least one pathogen that attacks livestock farms, in particular cattle and pigs. The DNA vaccine is improved either by its formulation or by the addition of GM-CSF or by the optimization of the antigen(s) or by a combination of the above.

The DNA vaccine is preferably improved by its formulation, or optionally by either the addition of GM-CSF, or the optimization of the antigen(s), or the addition of GM-CSF and the optimization of the antigen(s).

By definition, a DNA vaccine contains a gene or a gene fragment as an active ingredient, e.g. a plasmid encoding and expressing an epitope. A plasmid is a DNA transcription unit that contains a polynucleotide sequence that contains the sequence encoding the gene to be expressed and the elements necessary for in vivo expression. A supercoiled or otherwise circular plasmid form is preferred. The linear form is also within the scope of the invention.

Each plasmid contains a promoter that ensures that the gene introduced under its control is expressed in the host cell. Usually a strong eukaryotic promoter of human or murine origin and in particular the cytomegalovirus early CMV-IE promoter, or optionally a promoter from another source such as rat or guinea pig.

- 10 More generally, the promoter may be of viral or cellular origin. Viral promoters other than CMV-IE include the early or late promoter of SV40 virus or the LTR promoter of Rous Sarcoma virus. The promoter may also be the promoter of the virus from which the gene is derived, such as a promoter specific for the gene. A cellular promoter may include, for example, a cytoskeletal gene, such as the promoter for desmin or actin. If a plasmid contains many genes, these may be located in the same transcription unit or in different ones.

In one embodiment of the invention, the DNA vaccines of the invention are formulated to contain as an adjuvant a cationic lipid comprising a quaternary ammonium salt of the following general formula I:

CH ₃

Ri-O-CH ₂ -CH-CH ₂ -N ⁺ -r ₂ -x (I) II

ORi CH ₃ where in the formula R1 represents a saturated or unsaturated, straight-chain aliphatic group having 12-18 carbon atoms, _R2 represents another aliphatic group having 2-3 carbon atoms, and X represents a hydroxyl or amino group.

This is preferably DMRIE [N-(2-hydroxyl)-N,N-dimethyl-2,3bis(tetradecyloxy)-1-propaneammonium; WO-A-9634109], which is preferably combined with a neutral lipid, in particular DOPE.

- 11 (dioleoylphosphatidylethanolamine) combined, thus giving DMRIE-DOPÉ.

The invention therefore relates to a DNA vaccine against at least one pathogen that endangers domestic animals, in particular cattle and pigs, and which comprises at least one plasmid comprising at least one nucleotide sequence encoding an immunogen of a pathogen of the given animal species, under conditions which allow the in vivo expression of the sequence, and a quaternary ammonium salt cationic lipid, preferably DOPE, in particular in combination with DMRIE.

The recombinant vector is preferably mixed with the above adjuvant immediately before use, and it is advantageous to allow the resulting mixture to stand for, for example, 10-60 minutes, in particular for a period of the order of 30 minutes, to allow complex formation before administration to the animal.

When DOPE is used, the molar ratio of DMRIE to DOPE is preferably between 95:5 and 5:95, more preferably 1:1.

The weight ratio of plasmid to DMRIE or DMRIE-DOPE adjuvant is between 50:1 and 1:10, in particular between 10:1 and 1:5, preferably between 1:1 and 1:2.

In another embodiment of the invention, GM-CSF (granulocyte macrophage colony-stimulating factor; Grant S.M. et al., Drugs 53, 516 (1992)) is added to the vaccine of the invention; this can be achieved by adding the GMCSF protein directly to the vaccine composition or, preferably, by adding the nucleotide encoding GM-CSF.

- 12 sequences are introduced into an expression vector, under conditions that allow their expression in vivo. A plasmid may preferably be used as the expression vector, for example a plasmid containing the nucleotide sequence encoding the antigen(s) of interest or another plasmid. The GM-CSF is preferably selected taking into account the species of animal to be vaccinated; for example, bovine GM-CSF may be used for cattle; porcine GM-CSF may be used for pigs.

According to a third embodiment of the invention, the nucleotide sequence(s) encoding the immunogen are optimized. Optimization may mean any modification of the nucleotide sequence, in particular which results in at least higher expression of the nucleotide sequence and/or an increase in the stability of the messenger RNA encoding the antigen and/or the initiation of the secretion of the antigen into the extracellular medium, and which enhances the induced immune response as a direct or indirect effect.

In the context of the invention, the optimization of the antigen in question preferably comprises the following steps: deletion of a nucleotide sequence fragment encoding the transmembrane domain of the antigen in question (deletion means a complete or partial deletion of the transmembrane domain sufficient to render the transmembrane domain no longer or substantially no longer functional) and/or a nucleotide sequence fragment encoding the tPA signal [Montgomery et al., Cell. Mol. Biol. 43, 285-292 (1997); Harris et al., Mol. Biol. Med. 3, 279-292 (1986)]

- addition of 13 carriers, and/or insertion of a stabilizing intron 5' from the gene to be expressed. Deletion of the DNA fragment encoding the transmembrane domain of the antigen in question promotes the secretion of the thus truncated antigens into the extracellular medium, thereby increasing the probability that they will come into contact with cells of the immune system. Insertion of the nucleotide sequence encoding the tPA signal promotes the translatability of the messenger RNA linked to the tPA signal, thereby increasing the expression of the messenger RNA and thus the production of the antigens. The tpA signal also plays a role in the secretion of the synthesized antigens.

Other nucleotide sequences encoding signal peptides may also be used, in particular the signal peptide of mellitin obtained from bees [Sisk W.P. et al., J. Virol 68, 766-775 (1994)].

By inserting a stabilizing intron into the gene encoding the antigen in question, we can avoid abnormal splicing of its messenger RNA and maintain the physical integrity of the messenger RNA.

The tPA signal is preferably of human origin. The nucleotide sequence of the human tPA signal is available in the GenBank database under accession number NM_000930. The intron is preferably intron II of the rabbit beta-globin gene [van Ooyen et al., Science 206, 337-344 (1979)], the nucleotide sequence of which is available in the GenBank database under accession number V00882 and its intron reference number 2.

- 14 The invention relates to an improved DNA vaccine that induces an effective and protective immune response in cattle against infectious bovine rhinotracheitis (IBR).

The virus that causes infectious bovine rhinotracheitis is a bovine herpesvirus type 1 (BHV-1), a member of the Alphaherpesviridae family [Babiuk L.A. et al., Vet. Microbiol, 53, 31-42 (1996)]. The nucleotide sequences encoding the gB, gC and gD glycoproteins are known and can be found in the GenBank database under accession number AJ004801.

According to the invention, the DNA vaccine against IBR is preferably of improved formulation, as it contains an adjuvant according to the invention, in particular DMRIE, preferably DMRIE-DOPE. The composition may optionally further include bovine GM-CSF [Maliszewski et al., Molec. Immunol. 25, 843-850 (1988)], or may optimize at least one IBR antigen, or may add bovine GM-CSF and optimize at least one IBR antigen.

A nucleotide sequence encoding bovine GM-CSF is available in the GenBank database under accession number U22385.

The addition of bovine GM-CSF can be achieved by incorporating the bovine GM-CSF polypeptide into the vaccine composition, or preferably by inserting the nucleotide sequence encoding GM-CSF into an in vivo expression vector, preferably a plasmid. The nucleotide sequence encoding bovine GM-CSF is preferably a

- 15 are inserted into another expression plasmid (e.g. pLF1032; Id. Example 13), not the same one(s) into which the gene(s) encoding the IBR antigen(s) were inserted.

The optimization of IBR-derived antigens can be achieved by replacing the sequence of the signal peptide of the gB glycoprotein and/or the gC glycoprotein and/or the gD glycoprotein with a "signal sequence", in particular the human tPA signal (GenBank accession number: NM_000930), and/or deleting the DNA fragment encoding the transmembrane domain of gB and/or gC and/or gD. The DNA fragment encoding the transmembrane domain of one of the listed glycoproteins is preferably deleted together with the adjacent C-terminal part (the cytoplasmic part of the glycoprotein). The DNA vaccine against IBR according to the invention can therefore encode a single optimized IBR antigen (gB, gC or gD), or two of them, or all three, i.e. an optimized gB, an optimized gC and an optimized gD.

The nucleotide sequences encoding BHV-1 antigens that can be used in the invention, as well as the various expression vector constructs prepared therefrom, are described in the examples accompanying the description and in document FR-A1-2751229, in particular in Examples 7 and 8 and in Figures 3 and 4.

According to the invention, the DNA vaccine against BHV-1 is preferably formulated with DMRIE-DOPE and comprises an expression plasmid (e.g. pPB281, Example 3.1.2) encoding the gB antigen of BHV-1, which is optimized by deleting the transmembrane domain and the

- 16 adjacent nucleotide sequence fragments encoding the C-terminal part, and further comprising another expression plasmid (e.g. pPB292, Example 3.2.2) encoding the BHV-1 gAntigen, which is optimized by deleting the transmembrane domain and the adjacent C-terminal part encoding nucleotide sequence fragment, and comprising a third expression plasmid (e.g. pPB284, Example 3.3.2) encoding the BHV-1 gDantigen, which is optimized by deleting the transmembrane domain and the adjacent C-terminal part encoding nucleotide sequence fragment.

In general, and not only in the case of BHV-1, the C-terminal region adjacent to the transmembrane domain coding sequence can be retained. However, it is usually simpler to delete the transmembrane domain coding sequence at the same time.

The invention also provides an improved DNA vaccine that induces an effective and protective immune response in cattle against bovine respiratory syncytial virus (BRSV).

BRSV is a Paramyxovirus, a member of the Paramyxoviridae family [Baker et al., Vet. Clin. North Am. Food Tinim. Pract. 13, 425-454 (1997)]. The nucleotide sequences encoding the F protein and the G glycoprotein are known and are found in the GenBank database under accession numbers Y17970 and U33539, respectively.

- 17 The DNA vaccine against BRSV is preferably formulated with an adjuvant according to the invention, in particular DMRIE, preferably DMRIEDOPÉ. The composition may optionally include bovine GM-CSF, or may include at least one BRSV antigen, or may include bovine GM-CSF and may include at least one BRSV antigen.

The addition of bovine GM-CSF can be done in the same way as described for BHV-1.

The optimization of antigens derived from BRSV can be achieved by replacing the signal sequence of the BRSV F protein and/or the BRSV G envelope glycoprotein with a "signal sequence", in particular the human tPA signal, and/or deleting the DNA fragment encoding the transmembrane domain of F and/or G. The DNA fragment encoding the transmembrane domain of one of the listed proteins is preferably deleted together with the adjacent C-terminal part. The DNA vaccine against BRSV according to the invention can therefore encode a single optimized BRSV antigen (F or G) or both (F and G).

The nucleotide sequences encoding BRSV antigens that can be used in the invention, as well as the various expression vector constructs prepared therefrom, are described in the examples of the present specification, FR-A1-2751229, in particular in Examples 9 and 10, and in Figures 5 and 6.

According to the invention, the DNA vaccine against BRSV is preferably formulated with DMRIE-DOPE and comprises an expression plasmid (e.g. pSB114, Example 4.1.3) which

- 18 encodes the BRSV F-antigen, which is optimized by inserting the human tPA signal sequence in place of the F signal sequence and deleting the nucleotide sequence fragment encoding the transmembrane domain and the adjacent C-terminal part thereof, and further contains another expression plasmid (e.g. pSBHO, Example 4.2.2) which encodes the BRSV G-antigen, which is optimized by inserting the human tPA signal sequence in place of the G signal sequence and deleting the nucleotide sequence fragment encoding the transmembrane domain and the adjacent C-terminal part thereof.

The invention also provides an improved DNA vaccine that induces an effective and protective immune response against BVDV in cattle.

BVDV is a pestivirus of the Flaviviridae family. It is commonly found in cattle populations, causing embryonic deformities, abortion, or clinical respiratory signs (mucosal disease) and intestinal signs (bovine viral diarrhea).

BVDV viruses can be divided into two groups based on the severity of clinical signs - the first is the type 1 BVDV group (no visible or mild clinical signs), and the second is the type 2 BVDV group (acute clinical signs, bleeding, high morbidity, high mortality) [Dean H.J. and Leyh R., Vaccine 17, 1117-1124 (1999)].

If a BVDV virus is not clearly defined, it should be considered type 1 or 2.

- 19 BVDV is a single-stranded RNA virus with an envelope. It contains a single gene that encodes a polyprotein. The cleavage of the polyprotein produces several well-characterized proteins, such as the EO protein (gp48) and the E2 protein (gp53) [Vassilev V.B. et al., J. Virol. 71, 471-478 (1997)].

The nucleotide sequences encoding the E0-E2 polyproteins are known and can be found in the GenBank database under accession numbers M96687 for BVDV-1 and AF145967 for BVDV-2.

The DNA vaccine against BVDV is preferably formulated with an adjuvant according to the invention, in particular DMRIE, preferably DMRIEDOPÉ. The composition may optionally include bovine GM-CSF, or may include at least one BVDV antigen, or may include bovine GM-CSF and may include at least one BVDV antigen.

The addition of bovine GM-CSF can be done in the same way as described for BHV-1.

The optimization of antigens derived from BVDV can be achieved by adding a "signal sequence", in particular the human tPA signal, 5' to the nucleotide sequence encoding the BVDV EO protein and/or the BVDV E2 protein, and/or deleting the DNA fragment encoding the E2 transmembrane domain, and/or inserting an intron, in particular intron II of the rabbit beta-globin gene, 5' to the nucleotide sequence encoding EO and/or E2. The DNA vaccine against BVDV according to the invention can therefore encode a single optimized BVDV antigen (EO or E2), or both (EO and E2).

- 20 The nucleotide sequences encoding BVDV antigens that can be used in the invention, as well as the various expression vector constructs prepared therefrom, are described in the examples accompanying the description and in document FR-A1-2751229, in particular in Example 13 and Figure 9, respectively.

According to the invention, the DNA vaccine against BVDV is preferably formulated with DMRIE-DOPE and comprises an expression plasmid (e.g. pLF1029, Example 5.1.2; pLF1031, Example 6.2.2) encoding the EO antigen of BVDV, which is optimized to insert the human tPA signal sequence 5' from the EO and the rabbit beta-globin gene II 5' from the EO. intron, and also contains another expression plasmid (e.g. pLF1021, Example 5.2.2; pLF1023, Example 6.1.2) encoding the BVDV E2 antigen, which is optimized to insert the human tPA signal sequence 5' from E2, and intron II of the rabbit beta-globin gene 5' from E2.

Preferably, plasmid mixtures can also be prepared. The mixture can contain at least two plasmids encoding different immunogens (E0 or E2) and/or derived from different types of BVDV (BVDV-1 or BVDV-2). Particularly preferred is a mixture containing four plasmids expressing BVDV-1 EO, BVDV-1 E2, BVDV-2 EO and BVDV-2 E2.

- 21 The invention also provides an improved DNA vaccine that induces an effective and protective immune response in cattle against parainfluenza virus type 3 (bPI-3).

The bPI-3 virus is a Paramyxovirus, also a member of the Paramyxoviridae family [Tsai et al., Infect. Immun. 11, 783-803 (1975)].

The nucleotide sequences encoding the hemagglutinin and neuraminidase proteins (HN) and the fusion protein (F) of bPI-3 are known and can be found in the GenBank database under accession number U31671.

The DNA vaccine against bPI-3 is preferably formulated with an adjuvant according to the invention, in particular DMRIE, preferably DMRIEDOPÉ. The composition may optionally include bovine GM-CSF, or may include at least one optimized bPI-3 antigen, or may include bovine GM-CSF and may include at least one optimized bPI-3 antigen.

The addition of bovine GM-CSF can be done in the same way as described for BHV-1.

The optimization of antigens derived from bPI-3 can be achieved by replacing the signal sequence of the hemagglutinin-neuraminidase (HN) protein of bPI-3 and/or the bPI-3 fusion protein (F) with a "signal sequence", in particular the human tPA signal, and/or deleting the DNA fragment encoding the transmembrane domain of HN and/or F, and/or inserting an intron, in particular intron II of the rabbit beta-globin gene, 5' from the nucleotide sequence encoding HN and/or F. The listed

- The DNA fragment encoding the transmembrane domain of one of the proteins is preferably deleted together with the adjacent C-terminal part. The DNA vaccine against bPI-3 according to the invention may therefore encode a single optimized bPI-3 antigen (HN or F) or may encode both (HN and F).

The nucleotide sequences encoding bPI-3 antigens that can be used in the invention, as well as the various expression vector constructs prepared therefrom, are described in the examples of the present specification and in document FR-A1-2751229, in particular in Examples 14 and 15 and in Figures 10 and 11.

According to the invention, the DNA vaccine against bPI-3 is preferably formulated with DMRIE-DOPE and comprises an expression plasmid (e.g. pLF1025, Example 7.1.2) encoding the HN antigen of bPI-3, which is optimized by inserting the human tPA signal sequence in place of the HN signal sequence, deleting the nucleotide sequence fragment encoding the transmembrane domain and the adjacent C-terminal part, and inserting the rabbit beta-globin gene II in the 5' direction from the HN. intron, and also contains another expression plasmid (e.g. pLF1027, Example 7.2.2) encoding the bPI-3 Fantigen, which is optimized by inserting the human tPA signal sequence in place of the F signal sequence, deleting the nucleotide sequence fragment encoding the transmembrane domain and the adjacent C-terminal part, and the nucleotide sequence fragment 5' from the F

- Inserted in 23 directions across intron II of the rabbit beta-globin gene.

The invention relates to an improved DNA vaccine that induces an effective and protective immune response in pigs against infectious porcine herpesvirus (PRV).

PRV virus is a member of the Alphaherpesviridae family and is the causative agent of Aujeszky's disease [Sawitzky D., Arch. Virol. Suppl. 13, 201-206 (1997)].

The nucleotide sequences encoding the gB, gC and gD glycoproteins are known and can be found in the GenBank database under accession numbers M17321, AF158090 and AF086702.

According to the invention, the DNA vaccine against PRV is preferably of improved formulation, as it contains an adjuvant according to the invention, in particular DMRIE, preferably DMRIE-DOPE. The composition may optionally further include porcine GM-CSF [Inumaru S. and Takamatsu H., Immunol. Cell. Biol. 73, 474-476 (1995)], or at least one PRV antigen may be optimized, or porcine GM-CSF may be added and at least one PRV antigen may be optimized.

The addition of porcine GM-CSF can be achieved by incorporating the porcine GM-CSF polypeptide into the vaccine composition, or preferably by inserting a nucleotide sequence encoding porcine GM-CSF (e.g., GenBank accession number D21074) into an in vivo expression vector, preferably a plasmid. The nucleotide sequence encoding porcine GM-CSF is preferably inserted into another expression plasmid (e.g., pLF1033; Example 14 of Id.)

- 24 and not into the same gene(s) into which the gene(s) encoding the PRV antigen(s) was inserted.

The optimization of PRV-derived antigens can be achieved by replacing the sequence of the signal peptide of the gB glycoprotein and/or the gC glycoprotein and/or the gD glycoprotein with a "signal sequence", preferably the human tPA signal (GenBank accession number: NM_000930), and/or deleting the DNA fragment encoding the transmembrane domain of gB and/or gC and/or gD. The DNA fragment encoding the transmembrane domain of one of the listed glycoproteins is preferably deleted together with the adjacent C-terminal part. The DNA vaccine against PRV according to the invention can therefore encode a single optimized PRV antigen (gB, gC or gD), or two of them, or all three, i.e. an optimized gB, an optimized gC and an optimized gD.

The nucleotide sequences encoding PRV antigens that can be used in the invention, as well as the various expression vector constructs prepared therefrom, are described in the examples accompanying the description and in document FR-A1-2751224, in particular in Examples 8 and 9, and in Figures 3 and 5.

According to the invention, the DNA vaccine against PRV is preferably formulated with DMRIE-DOPE and comprises an expression plasmid (e.g. pSB102, Example 8.1.2) encoding the gB antigen of PRV, which is optimized by deleting the transmembrane domain and the adjacent region.

C-terminal part encoding nucleotide sequence fragment, and further comprises another expression plasmid (e.g. pSB104, Example 8.2.2) encoding the PRV gAntigen, which is optimized to delete the transmembrane domain and the nucleotide sequence fragment encoding the adjacent C-terminal part thereof, and comprises a third expression plasmid (e.g. pSB106, Example 8.3.2) encoding the PRV gDantigen, which is optimized to delete the transmembrane domain and the nucleotide sequence fragment encoding the adjacent C-terminal part thereof.

The invention relates to an improved DNA vaccine that induces an effective and protective immune response in pigs against infectious porcine reproductive respiratory syndrome virus (PRRSV).

PRRSV virus is an Arterivirus, a member of the Arteriviridae family [Murtaugh et al., Arch. Virol. 140, 14511460 (1995)].

The nucleotide sequences encoding the proteins encoded by the ORF3, ORF5 and ORF6 open reading frames are known and can be found in the GenBank database under accession numbers U87392.

According to the invention, the DNA vaccine against PRRSV is preferably of improved formulation, as it contains an adjuvant according to the invention, in particular DMRIE, preferably DMRIE-DOPE. The composition may optionally include porcine GM-CSF or may be optimized for at least one of the following:

- 26 PRRSV antigens, or we can add porcine GM-CSF and optimize at least one PRRSV antigen.

The addition of porcine GM-CSF can be done in the same way as for PRV.

The optimization of antigens derived from PRRSV can be achieved by replacing the sequence of the signal peptide of the protein encoded by the 3rd open reading frame (ORF3, gp45 or large envelope glycoprotein) and/or the ORF5 glycoprotein (gp25 or E envelope glycoprotein) and/or the ORF6 glycoprotein (gpl8 or membrane protein) with a "signal sequence", in particular the human tPA signal (GenBank accession number: NM_000930), and/or deleting the DNA fragment encoding the transmembrane domain of ORF3 and/or ORF5 and/or ORF6. The DNA fragment encoding the transmembrane domain of one of the listed glycoproteins is preferably deleted together with the adjacent C-terminal part. The DNA vaccine against PRRSV according to the invention can therefore encode a single optimized PRRSV antigen (ORF3, 0RF5 or 0RF6), or two of them, or all three, i.e. an optimized 0RF3, an optimized ORF5 and an optimized ORF6.

The nucleotide sequences encoding PRRSV antigens that can be used in the invention, as well as the various expression vector constructs prepared therefrom, are described in the examples accompanying the description and in document FR-A1-2751224, in particular in Examples 14-17 and Figures 14-17.

- 27 According to the invention, the DNA vaccine against PRRSV is preferably formulated with DMRIE-DOPE and comprises an expression plasmid (e.g. pLF1009, Example 9.1.1; pLF1015, Example 10.1.1) encoding the ORF3 antigen of PRRSV, comprises another expression plasmid (e.g. pLF1012, Example 9.2.2; pLF1018, Example 10.2.2) encoding the ORF5 antigen of PRRSV, which is optimized by replacing the signal sequence of ORF5 with the sequence of the human tPA signal peptide and deleting the nucleotide sequence fragment encoding the transmembrane domain and the adjacent C-terminal part, and comprises a third expression plasmid (e.g. pLF1014, Example 9.3.2; pLF1016, Example 10.3.2), which encodes the ORF6 antigen of PRRSV, which is optimized by replacing the signal sequence of ORF6 with the sequence of the human tPA signal peptide, and deleting the nucleotide sequence fragment encoding the transmembrane domain and the adjacent C-terminal part.

Preferably, plasmid mixtures can also be prepared. The mixture can contain at least two plasmids encoding different immunogens (ORF3, ORF5 or ORF6) and/or originating from different PRRSV strains (e.g., the European strain, e.g., Lelystad, the American strain ATCC VR-2322). Particularly preferred is a mixture containing six plasmids expressing PRRSV Lelystad ORF3, PRRSV Lelystad ORF5, PRRSV Lelystad ORF6, PRRSV VR-2332 ORF3, PRRSV VR-2332 ORF5 and PRRSV VR-2332 ORF6.

- 28 : .- .··,í • 4 ··« * * ««« _{V t} ♦··· · · · · · ····

The invention also provides an improved DNA vaccine that induces an effective and protective immune response in pigs against swine influenza virus type 3 (SIV).

SIV is a type A influenza virus, a member of the Orthomyxoviridae family [Murphy B.R. and Webster R.G., Virology, 2nd ed., eds. B.N. Fields, D.M. Knipe et al., Raven Press Ltd., New York (1990)].

The nucleotide sequences encoding the hemagglutinin (HA) and neuraminidase (NA) proteins of SIV H1N1 and H3N2 strains are known and can be found in the GenBank database under accession numbers K00992, U86145, U07146 and AF153238.

The SIV DNA vaccine is preferably formulated with an adjuvant according to the invention, in particular DMRIE, preferably DMRIEDOPE. The composition may optionally include porcine GM-CSF, or may include at least one SIV antigen, or may include porcine GM-CSF and may include at least one SIV antigen.

The addition of porcine GM-CSF can be done in the same way as for PRV.

SIV-derived antigens can be optimized by replacing the signal sequence of the SIV hemagglutinin protein (HA) and/or the SIV neuraminidase protein (NA) with a "signal sequence", in particular the human tPA signal, and/or deleting the DNA fragment encoding the transmembrane domain of HA and/or NA, and/or inserting an intron, in particular intron II of the rabbit betaglobin gene, 5' from the nucleotide sequence encoding HA and/or NA. One of the proteins listed

- 29 » ·* ·* ** · * * · * « « * • ν ·· « · ·*· « ' ♦··* · * · · ·«·« • ·· ·· ·« * The DNA fragment encoding the transmembrane domain is preferably deleted together with the adjacent C-terminal part. The DNA vaccine against SIV according to the invention may therefore encode a single optimized SIV antigen (HA or NA), or both (HA and NA).

The nucleotide sequences encoding SIV antigens that can be used in the invention, as well as the various expression vector constructs prepared therefrom, are described in the examples accompanying the description and in document FR-A1-2751224, in particular in the case of the SIV HIN1 strain in Examples 10 and 11, and in Figures 7 and 9, and in the case of the SIV H3N2 strain in Examples 12 and 13, and in Figures 11 and 13.

According to the invention, the DNA vaccine against SIV is preferably formulated with DMRIE-DOPE and comprises an expression plasmid (e.g. pLF1002, Example 11.1.2; pLF1006, Example 12.1.2) encoding the HA antigen of SIV, which is optimized by inserting the human tPA signal sequence in place of the HA signal sequence, deleting the nucleotide sequence fragment encoding the transmembrane domain and the adjacent C-terminal part, and inserting the rabbit betaglobin gene II in the 5' direction from the HA. intron, and also contains another expression plasmid (e.g. pLF1004, example 11.2.2; pLF1008, example 12.2.2) encoding the SIV NA antigen, which is optimized by inserting the human tPA signal sequence in place of the NA signal sequence, deleting the transmembrane domain and the adjacent Ci. £ J

- A nucleotide sequence fragment encoding 30 terminal parts and intron II of the rabbit betaglobin gene are inserted 5' from the NA.

Preferably, plasmid mixtures can also be prepared. The mixture can contain at least two plasmids encoding different immunogens (HA or NA) and/or derived from different SIV strains (e.g. H1N1 or H3N2). Particularly preferred is a mixture containing four plasmids expressing H1N1 HA, H1N1 NA, H3N2 HA and H3N2 NA.

Although the invention is described with respect to a specific DNA vaccine, the invention, and in particular the use of the adjuvants of the invention, also applies to DNA vaccines against other pathogens of the above animal species.

Following the same line of thought, the vaccines of the invention can also be combined with other and/or DNA vaccines against other pathogens of a given animal species.

Other such pathogens include, for example, rabies virus, swine cholera virus and porcine parvoviruses.

The immunogenic compositions or improved DNA vaccines against rabies virus according to the invention preferably comprise a plasmid encoding the unmodified G-glycoprotein of rabies virus, DMRIE-DOPE and optionally GM-CSF.

The improved immunogenic composition or DNA vaccine against porcine parvovirus according to the invention preferably comprises a plasmid comprising a vector of porcine parvovirus.

- 31-derived antigens (e.g. VP2 protein, Id. Example 18 and Figure 18 of document FR-A1-2751224), as well as DMRIE-DOPÉ and optionally porcine GM-CSF (e.g. pLF1033, Example 14).

The improved immunogenic composition or DNA vaccine against hog cholera virus (HCV) according to the invention preferably comprises a plasmid encoding an antigen derived from HCV (e.g. the E1 protein, see example 19 and figure 19 of document FR-A1-2751224; or the E2 protein, see example 20 and figure 20 of document FR-A1-2751224), DMRIE-DOPE and optionally porcine GM-CSF (e.g. pLF1033, example 14).

The invention therefore also provides multivalent DNA vaccines that can provide effective protection in cattle against at least two bovine pathogens selected from the following: BHV-1, BRSV, BVDV, bPI-3 and rabies viruses.

The invention therefore also provides multivalent DNA vaccines that can provide effective protection in pigs against at least two porcine pathogens selected from the following: PRV virus, PRRSV virus, SIV virus, hog cholera virus (or HCV) and porcine parvoviruses.

Multivalent DNA vaccines may be formulated in an improved manner by containing an adjuvant according to the invention, in particular DMRIE, preferably DMRIE-DOPE. The formulation may optionally further include GM-CSF (see above), or may be optimized for at least one of the adjuvants in question.

- 32 antigens (Id. above), or we can add GM-CSF and optimize at least one antigen of interest.

The multivalent DNA vaccines of the invention comprise one or more expression plasmids such that the vaccines express in vivo at least one immunogen against a first pathogen and at least one immunogen against a second pathogen, which pathogens infect the same animal species. Preferably, at least one of the immunogens is selected from the following:

- for cattle: BRSV F, BRSV G, BHV-1 gB, BHV-1 gC, BHV-1 gD, BVDV-1 EO, BVDV-1 E2, BVDV-2 EO, BVDV-2 E2, bPI-3 F and bPI-3 HN, and

- for pigs: PRV gB, PRV gC, PRV gD, PRRSV Lelystad strain 0RF3, PRRSV Lelystad strain 0RF5, PRRSV Lelystad strain ORF6, PRRSV VR-2332 strain 0RF3, PRRSV VR2332 strain ORF5, PRRSV VR-2332 strain 0RF6, SIV HINl strain HA, SIV HINl strain NA, SIV H3N2 strain HA, SIV H3N2 strain NA.

The improved monovalent or multivalent DNA vaccines of the invention can be combined with at least one conventional vaccine (inactivated vaccine, attenuated live vaccine, subunit vaccine) or with a recombinant vaccine using an in vivo expression vector (e.g. poxvirus, adenovirus, herpesvirus) that are active against at least one pathogen infecting the same animal species but different, or that can be used as a booster for such vaccines.

- 33 The skilled person can consult the document FR-A1-2751229 for the listed bovine valencies and FR-A1-2751224 for the listed porcine valencies for the methods for producing plasmids.

The invention also provides a method for vaccinating domestic animals, particularly cattle and pigs. The vaccination method comprises administering one of the described monovalent or multivalent improved DNA vaccines. Such vaccination methods may be used in pregnant females, or in young animals, or in adult animals, for passively transmitting immunity. The vaccination method comprises administering one or more doses of the improved DNA vaccine.

The amount of DNA in the vaccines of the invention is between about 10 and 1000 μg, preferably between about 50 and 500 μg, for a given plasmid. One skilled in the art has the expertise to accurately determine the effective dose of DNA to be used in each vaccination protocol.

The dose volume is preferably between about 0.2 and 5 ml, more preferably between 1 and 3 ml.

In this vaccination procedure, any of the various routes and methods of administration known in the art for polynucleotide vaccines can be used to administer the improved DNA vaccines of the invention.

According to a preferred embodiment of the invention, during vaccination procedures, the improved DNA vaccines of the invention can be administered intramuscularly, subcutaneously

- 34 route, or intradermally using a needle-free injector.

The invention will be described in more detail below with the help of embodiments disclosed in the examples, which do not limit the scope of protection.

Sequencer:

1.

ID number sequence:

PB326.

oligonucleotide number

2.

ID number sequence:

PB329.

oligonucleotide number

3.

ID number sequence:

SB090.

oligonucleotide sequence ID no.:

SB091.

oligonucleotide number

5.

same sequence number:

LF001.

oligonucleotide number

6.

ID number sequence:

LF002.

oligonucleotide sequence ID no.:

PB234.

oligonucleotide sequence ID no.:

PB235.

oligonucleotide number

9.

ID number sequence:

PB511.

oligonucleotide number

10.

ID number sequence:

PB512.

oligonucleotide number

11.

ID number sequence:

SB221.

oligonucleotide number

12.

ID number sequence:

SB222.

oligonucleotide number

13.

ID number sequence:

PB507.

oligonucleotide number .

ID number sequence:

PB508.

oligonucleotide number

15.

ID number sequence:

PB513.

oligonucleotide number

16.

ID number sequence:

PB514.

oligonucleotide number .

ID number sequence:

SB223.

oligonucleotide number

18.

ID number sequence:

SB224.

oligonucleotide number

19.

ID number sequence:

PB497.

oligonucleotide number

20.

ID number sequence:

PB498.

oligonucleotide number

21.

ID number sequence:

SB225.

oligonucleotide number

22.

ID number sequence:

SB226.

oligonucleotide number

SEQ ID NO: 23.

SEQ ID NO: 24.

SEQ ID NO: 25.

SEQ ID NO: 26.

SEQ ID NO: 27.

SEQ ID NO: 28.

SEQ ID NO: 29.

SEQ ID NO: 30.

SEQ ID NO: 31.

SEQ ID NO: 32.

SEQ ID NO: 33.

SEQ ID NO: 34.

SEQ ID NO: 35.

SEQ ID NO: 36.

SEQ ID NO: 37.

SEQ ID NO: 38.

SEQ ID NO: 39.

SEQ ID NO: 40.

SEQ ID NO: 41.

SEQ ID NO: 42.

SEQ ID NO: 43.

SEQ ID NO: 44.

SEQ ID NO: 45.

SEQ ID NO: 46.

SEQ ID NO: 47.

SEQ ID NO: 48.

SEQ ID NO: 49.

SEQ ID NO: 50.

SB210. No.

SB211. No.

SB212. No.

SB220. No.

SB213. No.

SB214. No.

SB215. No.

SB216. No.

No. LF050

No. LF051

No. LF052

No. LF053

No. LF039

No. LF040

No. LF041

No. LF042

No. LF043

No. LF044

No. LF045

No. LF046

No. LF064

No. LF065

No. LF066

No. LF067

No. LF047

No. LF048

No. LF058

LF059. Oligonucleotide Oligonucleotide Oligonucleotide Oligonucleotide Oligonucleotide Oligonucleotide Oligonucleotide Oligonucleotide Oligonucleotide Oligonucleotide Oligonucleotide Oligonucleotide Oligonucleotide Oligonucleotide Oligonucleotide Oligonucleotide Oligonucleotide

- 36 51.

.

53.

54.

55.

56.

.

58.

59.

60.

61.

62.

63.

.

65.

66.

67.

68.

69.

70.

71.

72.

73.

.

75.

76.

.

78.

ID No.: LF060. Oligonucleotide ID No.: LF061. Oligonucleotide ID No.: LF062. Oligonucleotide ID No.: LF063. Oligonucleotide ID No.: SB201. Oligonucleotide ID No.: SB202. Oligonucleotide ID No.: SB203. Oligonucleotide ID No.: SB217. Oligonucleotide ID No.: SB204. Oligonucleotide ID No.: SB205. Oligonucleotide ID No.: SB206. Oligonucleotide ID No.: SB218. Oligonucleotide ID No.: SB207. Oligonucleotide ID No.: SB208. Oligonucleotide ID No.: SB209. Oligonucleotide ID No.: SB219 Oligonucleotide ID No.: LF027 Oligonucleotide ID No.: LF028 Oligonucleotide ID No.: LF019 Oligonucleotide ID No.: LF020 Oligonucleotide ID No.: LF021 Oligonucleotide ID No.: LF022 Oligonucleotide ID No.: LF023 Oligonucleotide ID No.: LF024 Oligonucleotide ID No.: LF025 Oligonucleotide ID No.: LF026 Oligonucleotide ID No.: LF037 Oligonucleotide ID No.: LF038

ID number 79

ID number 80

ID number 81

ID number 82

ID number 83

ID number 84

ID number 85

ID number 86

ID number 87

ID number 88

ID number 89

ID number 90

ID number 91

ID number 92

ID number 93

ID number 94

ID number 95

ID number 96

ID number 97

ID number 98

ID number 99

ID number 100

ID number 101

ID number 102

ID number 103

ID number 104

ID number 105

SEQ ID NO: 106: LF029.

sequence: LF030.

sequence: LF031.

sequence: LF032.

sequence: LF033.

sequence: LF034.

sequence: LF035.

sequence: LF036.

Seq.: LF003.

Seq.: LF004.

Seq.: LF005.

Seq.: LF006.

Seq.: LF007.

Seq.: LF008.

sequence: LF009.

Seq.: LF010.

sequence: LF011.

sequence: LF012.

sequence: LF013.

sequence: LF014.

sequence: LF015.

sequence: LF016.

sequence: LF017.

sequence: LF018.

sequence: LF054.

sequence: LF055.

sequence: LF056.

sequence: LF057.

oligonucleotide number

- 38 Examples

For each pathogen tested and for each gene encoding the major antigens (native or modified), a specific construct was made in a eukaryotic expression plasmid. The secreted forms of the antigens were generated by deleting the gene fragments encoding the transmembrane and cytoplasmic domains. In each case, the transmembrane domain of the proteins was identified based on the hydrophobicity (“hydropathy”) profile of the corresponding protein sequence (generated in MacVector 6.5).

Example 1

Molecular biological procedures

1.1 Extraction of genomic DNA of viruses

The virus suspensions were treated with proteinase K (final concentration: 100 mg/ml) in the presence of sodium dodecyl sulfate (SDS) (final concentration: 0.5%) for 2 hours at 37°C. The viral DNA was then extracted using a phenol/chloroform mixture, precipitated with two volumes of absolute ethanol for 16 hours at -20°C, and centrifuged at 10,000xg for 15 minutes at 4°C. The DNA pellets were dried and then taken up in a minimal volume of sterile and ultrapure water.

1.2 Isolation of the viral genomic RNA

The genomic RNA of the viruses was extracted using the "guanidinium thiocyanate/phenol-chloroform method" of P. Chomczynsky and N. Sacchi [Tinái. Biochem. 162, 156-159 (1987)].

1.3 Molecular biological methods

All plasmid constructs were prepared using standard molecular biology methods [Id. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989)]. All restriction fragments used in the invention were isolated using the Geneclean reagent kit (BIO1O1 Inc., La Jolla, CA). The cloned DNA fragments and the junctions with the expression vector for each construct were sequenced using the Sanger method (Sambrook et al., 1989).

1.4 PCR and RT-PCR

Oligonucleotides specific for the cloned genes or gene fragments were synthesized, and in some cases some of these oligonucleotides contained restriction sites at their 5' ends to facilitate cloning of the amplified fragments. Reverse transcription (RT) reactions and polymerase chain reactions (PCR) were performed according to standard procedures (Sambrook et al., 1989).

1.5 Large-scale purification of plasmids

The purified plasmids used in the vaccine preparations were prepared in quantities of approximately 10 mg using the cesium chloride/ethidium bromide gradient method (Sambrook et al., 1989).

Example 2

A1ap plasmid constructs

Prepared from plasmid pVR1012 (see Figure 1 and Figure 1 and Example 7 of WO-A9803199)

- 40 pVR1020 eukaryotic expression plasmid [C.J., Luke et al., J. of Infectious Diseases 175, 95-97 (1997)] contains the frame encoding the signal sequence of human tissue plasminogen activator (tPA).

The pVR1020 plasmid is modified by BamHI-BglII digestion and insertion of a sequence containing multiple cloning sites (BamHI, NotI, EcoRI, XbaI, PmlI, PstI, BglII) and is prepared by annealing the following oligonucleotides:

PBR326 (40 members) (SEQ ID NO: 1)

5'-GATCTGCAGCACGTGTCTAGAGGATATCGAATTCGCGGCC-3' and PBR329 (40 members) (SEQ ID NO: 2)

5'-GATCCGCGGCGCGAATTCGATATCCTCTAGACAGGTGCT-3'.

The resulting vector, approximately 5105 base pairs (or bp) in length, is called pAB110 (Figure 2).

Intron II of the rabbit β-globin gene was cloned into the pCRII vector (Invitrogen, Carlsbad, CA, USA) after PCR generation of the corresponding DNA fragment using genomic DNA from rabbit peripheral blood cells as a template using the following oligonucleotides:

SB090 (20 members) (sequence ID 3)

5'-TTGGGGACCCTTGATTGTTC-3' and

SB091 (21 members) (SEQ ID NO: 4) 5'-CTGTAGGAAAAAGAAGAGGC-3'.

The expression plasmid pABHO is modified by inserting intron II of the rabbit globin gene into the SalI site located 5' from the ATG of the tissue plasminogen activator (tPA) signal peptide. Intron II of the rabbit globin gene.

- The sequence of intron 41 is amplified from the pNS050 plasmid by polymerase chain reaction (PCR) using the following oligonucleotide pair:

LF001 (30 members) (SEQ ID NO: 5) 5'-CTCCATGTCGACTTGGGGACCCTTGATTGT-3' and

LF002 (30 members) (SEQ ID NO: 6) 5'-CTCCATGTCGACCTGTAGGAAAAAGAAGAA-3'.

The resulting PCR product (573 base pairs or bp) is digested with SalI and then cloned into the previously linearized pABHO plasmid with SalI, resulting in the approximately 5678 bp plasmid pLF999.

Example 3

Plasmids encoding different forms of bovine herpesvirus type 1 (BHV-1) antigens

Viral DNA fragments containing the gB, gC and gD genes of BHV-1 strain B901 are isolated by digesting the viral genome with various restriction enzymes, separating the resulting fragments by agarose gel electrophoresis, and then analyzing them by Southern blotting using probes corresponding to fragments derived from the gB, gC and gD genes of BHV-1 ST strain [Leung-Tack P. et al. Virology 199, 409-421 (1994)]. BHV-1 Colorado strain (Cooper) (ATCC number: VR-864) can also be used. The fragments thus identified are cloned into the pBluescript SK+ vector (Stratagene, La Jolla, CA, USA), and the three genes are cloned from this into the pVR1012 expression vector.

3.1 Plasmids encoding different forms of BHV-1 gB

3.1.1 pPB280: gB gene cloned into pVR1012 vector (native form)

Two XhoI-XhoI fragments containing the 5' and 3' ends of the BHV-1 gB gene were identified by Southern blotting and cloned into XhoI-digested pBluescript SK+ vector (Stratagene, La Jolla, CA, USA). The resulting plasmids were named pPB128 and pPB117.

Plasmid pPB128 containing the 5' fragment of the gB gene was digested with NotI and XhoI to obtain a 1708 bp fragment (fragment A).

The plasmid pPB117 containing the 3' portion of the gB gene was digested with XhoI and StuI to obtain a 1345 bp fragment, which was cloned into the EcoRV and XhoI-digested pBluescript SK+ vector (Stratagene, La Jolla, CA, USA). The resulting plasmid was named pPB279. The pPB279 plasmid was then digested with XhoI and BamHI to obtain a 1413 bp DNA fragment (Bfragment).

The A and B fragments were cloned into the pBluescript SK+ vector digested with NotI and BamHI to obtain the plasmid pPB278 (approximately 6063 bp in length), which allowed the reconstitution of the BHV-1 gB gene.

The pPB278 plasmid was then used as a template for a PCR reaction using the following oligonucleotides:

- 43 PB234 (30 members) (sequence ID 7)

5'-TTGTCGACATGGCCGCTCGCGGCGGTGCTG-3' and

PB235 (21 members) (SEQ ID NO: 8) 5'-GCAGGGCAGCGGCTAGCGCGG-3'.

The resulting PCR product (146 bp) was digested with restriction enzymes SalI and NheI.

Plasmid PB278 was then digested with NheI and BamHI. The 2728 bp fragment thus obtained and the PCR fragment digested as above were ligated into the pVR1012 vector previously digested with SalI and BamHI (Example 2) to obtain plasmid pPB280 of approximately 7742 bp in length.

The gB gene of BHV-1 encodes a protein of 933 amino acids in length.

3.1.2 pPB281: gB gene cloned into pVR1012 vector (form A[TM-Cter])

A truncated form of the BHV-1 gB gene [from which the transmembrane (TM) domain and the carboxy-terminal (Cter) domain were deleted] was prepared by ligating a 2234 bp fragment obtained by SalI-PvuII digestion of plasmid pPB280 (Example 3.1.1) and a 56 bp fragment prepared by pairing the following oligonucleotides:

PB511 (52 members) (SEQ ID NO: 9) 5'CTGCACGAGCTCCGGGTTCTACGACATTGACCGCGTGGTCAAGACGGACTGA

G-3' and

- 44 PB512 (57 members) (SEQ ID NO: 10) 5'GATCCTCAGTCCGTCTTGACCACGCGGTCAATGTCGTAGAACCGGAGCTCG

TGCAG-3'

The resulting plasmid, approximately 7154 bp in length, was named pPB281. The truncated gB gene of BHV-1 encodes a protein of 759 amino acids in length.

3.1.3 pSB115: gB gene cloned into pABHO vector (tPA A[TM-Cter] form)

The tPA Á[TM-Cter] form of the BHV-1 gB gene was produced by PCR amplification of the pPB281 template (Example 3.1.2) using the following primers:

SB221 (39 members) (SEQ ID NO: 11) 5'-AAAATTTCGATATCCGCCGCGGGCGACCGGCGACAACG-3' and SB222 (33 members) (SEQ ID NO: 12) 5'-GGAAGATCTTCAGTCCGTCTTGACCACGCGGTC-3'.

The amplification product (2088 bp) was digested with EcoRV and BglII enzymes and then cloned into the pABHO vector (Example 2) previously digested with EcoRV and BglII, resulting in the plasmid pSB115 of approximately 7154 bp in length.

The tPA λ[TM-Cter] form of the gB gene encodes a 729 amino acid glycoprotein that contains the extracellular domain of the gB glycoprotein of BHV-1.

3.2 Plasmids encoding different forms of BHV-1 gC

3.2.1 pPB264: gC gene cloned into pVRl012 vector (native form)

A BamHIHindII fragment containing the full-length gC gene of BHV-1 was identified by Southern blotting and cloned into the pBluescript SK+ vector. The resulting plasmid was named pPB287.

The pPB287 plasmid was then digested with NcoI and BssSI to obtain a 1492 bp fragment, which was ligated together with a synthetic DNA fragment obtained by pairing the following oligonucleotides into pLitmus 39 plasmid (New England Biolabs, Inc., Beverly, MA, USA) previously digested with NcoI and XbaI:

PB507 (37 members) (SEQ ID NO: 13) 5'-TCGTGCCTGCGGCGCAAGGCCCGGGCGCGCCTGTAGT-3' and PB508 (37 members) (SEQ ID NO: 14) 5'-CTAGACTACAGGCGCGCCCGGGCCTTGCGCCGCAGGC-3', resulting in the intermediate plasmid pPB290.

pPB290 was digested with PstI and XbaI to obtain a 1554 bp fragment, which was cloned into the pVR1012 vector (Example 2) previously digested with PstI and XbaI. This resulted in the plasmid pPB264, which was approximately 6427 bp long. The gC gene of BHV-1 encodes a protein of 508 amino acids.

3.2.2 pPB292: gC gene cloned into pVR1012 vector (A[TM-Cter] form)

The truncated form of the BHV-1 gC gene was prepared by ligating the following three fragments into the pVR1012 vector (Example 2) previously digested with PstI and XbaI:

(a) a 1035 bp fragment obtained by digestion of pPB264 (Example 3.2.1) with PstI and XhoI, (b) a 350 bp fragment obtained by digestion of pPB264 with XhoI and BanI, (c) a 43 bp synthetic fragment obtained by annealing oligonucleotides PB513 and PB514.

The oligonucleotides mentioned are:

PB513 (43 members) (SEQ ID NO: 15)

5'-GCACCGCTGCCCGAGTTCTCCGGCAGCGCACGTACGACTAGT-3' and

PB514 (43 members) (SEQ ID NO: 16) 5'-CTAGACTAGTCGTACGTGGCGGTCGCGGAGAACTCGGGCAGCG-3'.

The resulting plasmid, approximately 6305 bp long, was named pPB292. The truncated gC gene of BHV-1 encodes a protein of 466 amino acids.

3.2.3 pSB116: gC gene cloned into pABl10 vector (tPA A[TM-Cter] form)

The tPA A[TM-Cter] form of the BHV-1 gC gene was produced by PCR amplification of the pPB292 template (Example 3.2.2) using the following primers:

SB223 (39 members) (SEQ ID NO: 17) 5'-AAAATTTCGATATCCCGGCGGGGGCTCGCCGAGGAGCG-3' and

- 47 SB224 (32 members) (SEQ ID NO: 18) 5'-GGAAGATCTCTAGTCGTACGTGGCGGTCGCGG-3'.

The amplification product (1362 bp) was digested with EcoRV and BglII enzymes and then cloned into the pAB110 vector (Example 2) previously digested with EcoRV and BglII, resulting in the plasmid pSB116 of approximately 6404 bp in length.

The tPA A[TM-Cter] form of the gC gene encodes a 479 amino acid glycoprotein that contains the extracellular domain of the BHV-1 gC glycoprotein.

3.3 Plasmids encoding different forms of BHV-1 gD

3.3.1 pPB148: gD gene cloned into pVR1012 vector (native form)

A 5 kb XhoI-XhoI fragment containing the BHV-1 gP gene was identified by Southern blotting and cloned into the XhoI-digested pBluescript SK+ vector. The resulting plasmid was named pPB147.

The plasmid pPB147 was digested with NdeI and BsrBI to give a 325 bp fragment, and digested with NdeI and Styl to give a 943 bp fragment, which was ligated into the pVR1012 vector previously digested with EcoRV and XbaI (Example 2). This gave the plasmid pPB148, which was approximately 6171 bp long. The gP gene of BHV-1 encodes a protein of 417 amino acids.

3.3.2 pPB284: gD gene cloned into pVR1012 vector (A[TM-Cter] form)

The truncated form of the BHV-1 gD gene was produced by PCR using genomic DNA of the BHV-1 virus strain B901, previously digested with PstI and XbaI, as a template, and the following primer pair:

PB497 (33 members) (SEQ ID NO: 19) '-TTTCTGCAGATGCAAGGGCCGACATTGGCCGTG-3' and

PB498 (31 members) (SEQ ID NO: 20) 5'-TTTCTAGATTAGGGCGTAGCGGGGGCGGGCG-3'.

The PCR fragment thus obtained was then cloned into the pVR1012 plasmid previously digested with PstI and XbaI (Example 2) to obtain the plasmid pPB284, which is approximately 5943 bp long. The truncated gD gene of BHV-1 encodes a protein of 355 amino acids.

3.3.3 pSB117: gD gene cloned into pABHO vector (tPA A[TM-Cter] form)

The tPA A[TM-Cter] form of the BHV-1 gD gene was produced by PCR amplification of the pPB284 template (Example 3.3.2) using the following primers:

SB225 (39 members) (SEQ ID NO: 21)

5'-AAAATTTCGATATCCCCGCGCCGCGGGTGACGGTATAC-3' and

SB226 (33 members) (SEQ ID NO: 22) 5'-GGAAGATCTTTAGGGCGTAGCGGGGGCGGGCGG-3'.

The amplification product (1029 bp) was digested with EcoRV and BglII enzymes and then cloned into the pABHO vector (Example 2) previously digested with EcoRV and BglII, which

- 49 resulted in the plasmid pSB117, which is approximately 6071 bp long.

The tPA A[TM-Cter] form of the gC gene encodes a 368 amino acid glycoprotein that contains the extracellular domain of the gD glycoprotein of BHV-1.

Example 4

Plasmids encoding different forms of bovine respiratory syncytial virus (BRSV) antigens

The genes encoding the F and G antigens of BRSV were prepared by RTPCR from viral RNA of the Snook strain [Thomas et al., Research in Vet. Science 22' 170-182 (1982)]. The BRSV strain A-51908 (ATCC number: VR794) can also be used.

4.1 Plasmids encoding different forms of BRSV-F

4.1.1 pSB107: F gene cloned into pVRl012 vector (native form)

The F gene of the BRSV Snook strain was amplified by RT-PCR using the following primers, using the viral RNA as a template:

SB210 (34 members) (SEQ ID NO: 23) '-AAATTTTCTGCAGATGGCCACAACAGCCATGAGG-3' and

SB211 (35 members) (SEQ ID NO: 24) 5'-TTAAGGATCCTCATTTACTAAGGAAAGATTGTTG-3'.

The 1739 bp amplification product was digested with PstI and BamHI enzymes and then cloned into the pVR1012 vector (Example 2) previously digested with PstI and BamHI, resulting in the approximately 6583 bp plasmid pSB107.

- 50 The F gene of BRSV encodes a protein of 574 amino acids in length.

4.1.2 pSB108: F gene cloned into pVR1012 vector (A[TMCter] form)

The truncated F gene of the BRSV Snook strain was amplified by RT-PCR using the following primers, using the viral RNA as a template:

SB210 (SEQ ID NO: 23) and

SB212 (39 members) (SEQ ID NO: 25) 5'-AATTTTGGATCCTCCATGTGGTGGATTTTCCTACATCTAC-3'.

The amplification product (1581 bp) was digested with PstI and BamHI enzymes and then cloned into the pVR1012 vector (Example 2) previously digested with PstI and BamHI, resulting in the plasmid pSB108 of approximately 6430 bp in length.

The truncated form of the F gene encodes a 523 amino acid protein that contains the extracellular domain of the BRSV Fglycoprotein.

4.1.3 pSB114: F-gene cloned into pABHO vector (tPA A[TM-Cter] form)

The tPA A[TM-Cter] form of the F gene of the BRSV Snook strain was amplified by RT-PCR using the following primers, using the viral RNA as a template:

SB212 (SEQ ID NO: 25) and

SB220 (38 members) (SEQ ID NO: 26) ' -AAAATTCACGTGAACATAACAGAAGAATTTTATCAATC-3 ' .

- 51 The amplification product (1516 bp) was digested with PmlI and BglII enzymes and then cloned into the pABHO vector (Example 2) previously digested with PmlI and BglII, resulting in the plasmid pSB114 of approximately 6572 bp in length.

The tPA λ[TM-Cter] form of the F gene encodes a 535 amino acid long protein containing the extracellular domain of the BRSV Fglycoprotein.

.2 Plasmids encoding different forms of BRSV-G

In the case of the BRSV G protein (type II glycoprotein), the signal sequence and the transmembrane sequence cannot be separated, so when the transmembrane domain is deleted, a signal sequence must be added 5’ from the sequence corresponding to the extracellular domain.

The pABHO plasmid (Example 2) was used to produce plasmids containing truncated forms of the gene encoding the BRSV G protein.

4.2.1 pSB109: G gene cloned into pVR1012 vector (native form)

The G gene of the BRSV Snook strain was amplified by RT-PCR using the following primers, using the viral RNA as a template:

SB213 (32 members) (SEQ ID NO: 27) 5'-ACCGCGTCGACATGTCCAACCATACCCATCATC-3' and

SB214 (38 members) (SEQ ID NO: 28) 5'-TTAAAATCTAGATTAGATCTGTGTAGTTGATTGATTTG-3'.

The amplification product (784 bp) was digested with SalI and XbaI enzymes, and then previously digested with SalI and XbaI.

- 52 was cloned into the digested pVR1012 vector (Example 2), resulting in the plasmid pSB109 of approximately 5661 bp in length.

The G gene of BRSV encodes a protein of 257 amino acids in length.

4.2.2 pSBHO: F-gene cloned into pABHO vector (tPA A[TM-Cter] form)

The truncated G gene of the BRSV Snook strain was amplified by RT-PCR using the following primers, using the viral RNA as a template:

SB215 (33 members) (SEQ ID NO: 29) .-TTTTAAGGATCCGCTAAAGCCAAGCCCACATCC-3' and

SB216 (33 members) (SEQ ID NO: 30) ' -TTAAAATCTAGATTAGATCTGTGTAGTTGATTG-3' .

The amplification product (666 bp) was digested with BamHI and XbaI enzymes and then cloned into the pAB110 vector (Example 2) previously digested with BamHI and XbaI, resulting in the plasmid pSB10 of approximately 5660 bp in length.

The tPA A[TM-Cter] form of the G gene encodes a 218 amino acid long protein that contains the extracellular domain of the BRSV Gglycoprotein, preceded by the signal sequence of the tissue plasminogen activator.

Example 5

Plasmids encoding different forms of bovine viral diarrhea virus type 1 (BVDV-1) antigens

The genes encoding the EO antigen (48 kDa glycoprotein or gp48) and the E2 antigen (gp53) of BVDV type 1 viruses were prepared by RT-PCR from viral RNA of the Osloss strain [L. De Moerlooze et al., J. Gen. Virol. 21, 1433-1438 (1993); A. Renard et al., DNA 4, 439-438 (1985); A. Renard et al., Ann. Rech. Vet. 18, 121-125 (1987)]. The NADL strain (ATCC No. VR-534) or the New York strain (ATCC No. VR-524) can also be used.

5.1 Plasmids encoding different forms of EO from the Osloss strain of BVDV type 1

5.1.1 pLF1028: EO gene cloned into pVR1012 vector (native form)

The complementary DNA (eDNA) of the EO gene of the Osloss strain was synthesized based on the corresponding viral RNA using primer LF051 and then amplified by PCR using the following oligonucleotide pair:

LF050 (36 members) (SEQ ID NO: 31) 5'-CATACCGTCGACATGAAGAAACTAGAGAAGCCCTG-3' and LF051 (40 members) (SEQ ID NO: 32) 5'-CATACCGGATCCTCAGGCTGCATATGCCCAAACCATGTC-3'. The approximately 765 bp DNA fragment obtained by digesting the PCR product with SalI and BamHI was ligated to the 4866 bp fragment obtained by digesting pVR1012 (Example 2) with SalI and BamHI,

- 54 resulting in the plasmid pLF1028 (approximately 5636 bp in length). The EO gene from the BVDV-1 Osloss strain encodes a protein of 252 amino acids in length.

An ATG codon was introduced into the sequence of oligonucleotide No. LF050, which allows for the initiation of translation of the corresponding recombinant EO polypeptide.

5.1.2 pLF1029: EO gene cloned into pLF999 vector (βglobin tPA-Εθ form)

The EO gene was synthesized by PCR reaction from the pLF1028 template (Example 5.1.1) using the following oligonucleotide pair:

LF052 (39 members) (SEQ ID NO: 33) '-CATGACGCGGCCGCTATGAAGAAACTAGAGAAAGCCCTG-3' and

LF053 (40 members) (SEQ ID NO: 34) 5'-CATGACAGATCTTTAGGCTGCATATGCCCCAAACCATGTC-3'.

The approximately 770 bp DNA fragment obtained by digesting the PCR product with NotI and BglII was ligated to the 5642 bp fragment obtained by digesting pLF999 (Example 2) with NotI and BglII, resulting in plasmid pLF1029 (approximately 6417 bp in length).

The modified EO gene (β-globin tPA-Εθ) from the Oslos strain of BVDV-1 encodes a protein of 283 amino acids.

5.2 Plasmids encoding different forms of E2 from the Osloss strain of BVDV type 1

5.2.1 pLF1020: E2 gene cloned into pVR1012 vector (native form)

The cDNA of the E2 gene of the Osloss strain was synthesized based on the corresponding viral RNA using primer LF040 and then amplified by PCR using the following oligonucleotide pair:

LF039 (33 members) (SEQ ID NO: 35) 5’-CATGACGTCGACATGACGACTACTGCATTCCTG-3’ and LF051 (40 members) (SEQ ID NO: 32) 5’-CATGACAGATCTTCAACGTCCCGAGGTCATTTGTTC-3’.

The approximately 1235 bp DNA fragment obtained by digesting the PCR product with SalI and BglII was ligated to the 4860 bp fragment obtained by digesting pVR1012 (Example 2) with SalI and BglII, resulting in plasmid pLF1020 (approximately 6100 bp in length).

The E2 gene from the Oslos strain of BVDV-1 encodes a protein of 409 amino acids in length.

An ATG codon was introduced into the sequence of oligonucleotide LF039, which allows for the initiation of translation of the corresponding recombinant E2 polypeptide.

5.2.2 pLF1021; E2 gene cloned into pLF999 vector (βglobin tPA-E2 A[TM+Cter] form)

The E2 gene deleted for the transmembrane and carboxy-terminal domains was synthesized by PCR reaction into pLF1020.

- 56 of the therapies (example 5.2.1), using the following oligonucleotide pair:

LF041 (36 members) (SEQ ID NO: 37) 5'-CATGACGCGGCCGCTATGACGACTACTGCATTCCTG-3' and LF042 (35 members) (SEQ ID NO: 38) 5'-CATGACAGATCTCCAAGCGAAGTAATCCCGGTGGTG-3'.

The approximately 1132 bp DNA fragment obtained by digesting the PCR product with NotI and BglII was ligated to the 5642 bp fragment obtained by digesting pLF999 (Example 2) with NotI and BglII, resulting in plasmid pLF1021 (approximately 6779 bp in length).

The modified E2 gene (β-globin tPA-E2 Á[TM+Cter]) from the Oslos strain of BVDV-1 encodes a protein of 404 amino acids.

Example 6

Plasmids encoding different forms of bovine viral diarrhea virus type 2 (BVDV-2) antigens

The genes encoding the E2 antigen (gp53) of BVDV type 2 viruses were prepared by RT-PCR from the viral RNA of strain 890 [J.F. Ridpath and S.R. Bolin, Virology 212, 36-46 (1995)]. Strain Q140, available from the Armand-Frappier Institute of the Ministry of Agriculture, Fisheries and Food of Quebec [P. Tijssen et al., Virology 217, 356-361 (1996)], can also be used. Strains 1373 and 296 can also be used (J.F. Ridpath, BVDV Research Project, National Animal Disease Center, 2300 Dayton Avenue, Ames, USA).

6.1 Plasmids encoding different forms of E2 from type 2 strain 890

6.1.1 pLF1022: E2 gene cloned into pVR1012 vector (native form)

The cDNA of the E2 gene of strain 890 was synthesized based on the corresponding viral RNA using primer LF044 and then amplified by PCR using the following oligonucleotide pair:

LF043 (36 members) (SEQ ID NO: 39) 5'-ACTGTATCTAGAATGACCACCACACAGCTTTCCTAATC-3' and LF044 (39 members) (SEQ ID NO: 40) 5'-ACTGTAAGATCTTTAAGTATTCACTCCAGCACCCATAGC-3'.

The approximately 1240 bp DNA fragment obtained by digesting the PCR product with XbaI and BglII was ligated to the 4891 bp fragment obtained by digesting pVR1012 (Example 2) with XbaI and BglII, resulting in plasmid pLF1022 (approximately 6136 bp in length).

The E2 gene from BVDV-2 strain 890 encodes a protein of 410 amino acids in length.

An ATG codon was introduced into the sequence of oligonucleotide No. LF043, which allows for the initiation of translation of the corresponding recombinant E2 polypeptide.

6.1.2 pLF1023: E2 gene cloned into pLF999 vector (βglobin tPA-E2 A[TM+Cter] form)

The E2 gene deleted for the transmembrane and carboxy-terminal domains was synthesized by PCR reaction from the pLF1022-58 template (Example 6.1.1) using the following oligonucleotide pair:

LF045 (41 members) (SEQ ID NO: 41) 5'-CATGACGCGGCCGCCCTATGACCACCACAGCTTTCCTAATC-3' and LF046 (36 members) (SEQ ID NO: 42) 5'-CATGACAGATCTTTTATATGAACTCTGAGAAGTAGTC-3'.

The approximately 1140 bp DNA fragment obtained by digesting the PCR product with NotI and BglII was ligated to the 5642 bp fragment obtained by digesting pLF999 (Example 2) with NotI and BglII, resulting in plasmid pLF1023 (approximately 6787 bp in length).

The modified E2 gene (β-globin tPA-E2 Δ[TM+Cter]) from BVDV-2 strain 890 encodes a protein of 405 amino acids.

6.2 Plasmids encoding different forms of EO from type 2 strain 890

6.2.1 pLF1030: EO gene cloned into pVR1012 vector (native form)

The cDNA of the EO gene of strain 890 was synthesized based on the corresponding viral RNA using primer LF065 and then amplified by PCR using the following oligonucleotide pair:

LF064 (39 members) (SEQ ID NO: 43) '-CATACCGTCGACATGAGAAAGAATTGGAGAAGGCACTG-3' and

LF065 (39 members) (SEQ ID NO: 44) 5'-CATACCGGATCCTCATGCTGCATGAGCACCAAACCATGC-3'.

- 59 The approximately 768 bp DNA fragment obtained by digesting the PCR product with SalI and BamHI was ligated to the 4866 bp fragment obtained by digesting pVR1012 (Example 2) with SalI and BamHI, resulting in plasmid pLF1030 (approximately 5639 bp in length). The EO gene from BVDV-2 strain 890 encodes a protein of 253 amino acids.

An ATG codon was introduced into the sequence of oligonucleotide No. LF064, which allows for the initiation of translation of the corresponding recombinant EO polypeptide.

6.2.2 pLF1031: EO gene cloned into pLF999 vector (βglobin tPA-Εθ form)

The EO gene was synthesized by PCR reaction from the pLF1030 template (Example 6.2.1) using the following oligonucleotide pair:

LF066 (42 members) (SEQ ID NO: 45)

5'-CATGACGCGGCCGCTATGAGAAGAAGAATTGGAGAAGGCACTG-3' and LF067 (39 members) (SEQ ID NO: 46) 5'-CATACCAGATCTTCATGCTGCATGAGCACCAAACCATGC-3'.

The approximately 770 bp DNA fragment obtained by digesting the PCR product with NotI and BglII was ligated to the 5642 bp fragment obtained by digesting pLF999 (Example 2) with NotI and BglII, resulting in plasmid pLF1031 (approximately 6417 bp in length).

The thus modified EO gene (β-globin tPA-Εθ) from BVDV-2 strain 890 encodes a protein of 283 amino acids.

Example 7

Plasmids encoding different forms of bovine parainfluenza virus type 3 (bPI-3) antigens

The genes encoding the hemagglutinin-neuraminidase (HN) and fusion (F) antigens of bPI-3 virus were prepared by RT-PCR from the viral RNA of the Reisinger SF-4 strain (available under ATCC number VR-281).

7.1. Plasmids encoding different forms of HN from the bPI-3 SF-4 strain

7.1.1 pLF1024: HN gene cloned into pVRl012 vector (native form)

The cDNA of the HN gene of the SF-4 strain was synthesized based on the corresponding viral RNA using primer LF048 and then amplified by PCR using the following oligonucleotide pair:

LF047 (39 members) (SEQ ID NO: 47) 5'-CATATCGTCGACATGGAATATTGGAAACACACAAACAGC-3' and LF048 (38 members) (SEQ ID NO: 48) 5'-CATGACGATATCTAGCTGCAGTTTTTCGGAACTTCTGT-3'.

The approximately 1726 bp DNA fragment obtained by digesting the PCR product with SalI and EcoRV was ligated to the 4896 bp fragment obtained by digesting pVR1012 (Example 2) with SalI and EcoRV, resulting in plasmid pLF1024 (approximately 6619 bp in length).

The HN gene of bPI-3 encodes a protein of 572 amino acids in length.

7.1.2 pLF1025: HN gene cloned into pLF999 vector (βglobin tPA-HN Δ[ΤΜ] form)

The HN gene deleted for the transmembrane domain was synthesized by PCR reaction from the pLF1024 template (Example 7.1.1) using the following oligonucleotide pair:

LF058 (33 members) (SEQ ID NO: 49)

5'-CATACTGCGGCCGCTTTAATTCAAGAGAACAAT-3' and

LF059 (35 members) (SEQ ID NO: 50) 5'-CATATCGATATCTAGCTGCAGTTTTTCGGAACTTC-3'.

The approximately 1566 bp DNA fragment obtained by digesting the PCR product with NotI and EcoRV was ligated to the 5663 bp fragment obtained by digesting pLF999 (Example 2) with NotI and EcoRV, resulting in plasmid pLF1025 (approximately 7229 bp in length).

The modified HN gene of bPI-3 (β-globin tPA-HN Δ[ΤΜ]) encodes a protein of 548 amino acids.

7.2. Plasmids encoding different forms of F from the SF-4 strain of bPI-3

7.2.1 pLF1026: F gene cloned into pVR1012 vector (native form)

The cDNA of the F gene of the SF-4 strain was synthesized based on the corresponding viral RNA using primer LF061 and then amplified by PCR using the following oligonucleotide pair:

LF060 (36 members) (sequence ID 51)

5'-CATATCGTCGACATGATCATCACAAACACAATCATA-3' and

LF061 (36 members) (SEQ ID NO: 52) ' -CATGACCAGATCTTTATTGTCTATTTGTCAGTATATA-3 ' .

The approximately 1628 bp DNA fragment obtained by digesting the PCR product with SalI and BglII was ligated to the 4860 bp fragment obtained by digesting pVR1012 (Example 2) with SalI and BglII, resulting in plasmid pLF1026 (approximately 6488 bp in length).

The F gene of bPI-3 encodes a protein of 550 amino acids in length.

7.2.2 pLF1027: F-gene cloned into pLF999 vector (βglobin tPA-F A[TM+Cter] form)

The F gene deleted for the transmembrane and C-terminal domains was synthesized by PCR reaction from the pLF1026 template (Example 7.2.1) using the following oligonucleotide pair:

LF062 (42 members) (SEQ ID NO: 53) ' -CATACTGCGGCCGCTCAAATAGACATAACAAAACTGCAACGT-3 ' and LF063 (41 members) (SEQ ID NO: 54) 5 ' -CATATCGATATCTATGCACTAGATTGATACCAACTTCCAAC-3 ' .

The approximately 1434 bp DNA fragment obtained by digesting the PCR product with NotI and EcoRV was ligated to the 5663 bp fragment obtained by digesting pLF999 (Example 2) with NotI and EcoRV, resulting in plasmid pLF1027 (approximately 7097 bp in length).

The thus modified F-gene of bPI-3 (β-globin tPA-F A[TM+Cter]) encodes a protein of 504 amino acids.

Example 8

Plasmids encoding different forms of pseudorabies virus (PRV) antigens

The genes encoding the gB, gC and gD glycoproteins of PRV were prepared by PCR from viral DNA of strain NIA3 [M. Riviére et al., J. Virol. 66, 3424-3434; A. Baskerville et al., The Veterinary Bulletin 43(9) (1973)]. Mutants of the NIA3 strain of PRV can also be used; Id. in U.S. Patent No. US-A-4,680,176, deposited under numbers 1-351 and 1-352 in the National Collection of Microorganism Cultures (CNCM), Pasteur Institute, Paris, France.

8.1 Plasmids encoding different forms of PRV-gB

8.1.1 pSB101: gB gene cloned into pVR1012 vector (native form)

The gB gene of PRV strain NIA3 was amplified by PCR using the following primers, using the viral DNA as a template:

SB201 (36 members) (SEQ ID NO: 55) 5'-TTTTAAGATATCATGCCCGCTGGTGGCGGTCTTTTGG -3' and SB202 (39 members) (SEQ ID NO: 56) 5'-TTTTAAGGATCCCTACAGGGCGTCGGGGTCCTCGCTCTC-3'.

The amplification product (2766 bp) was digested with EcoRV and BamHI enzymes and then cloned into the pVR1012 vector (Example 2) previously digested with EcoRV and BamHI, resulting in the plasmid pSB101 of approximately 7631 bp in length.

- 64 The PRV gB gene encodes a glycoprotein of 913 amino acids in length.

8.1.2 pSB102: gB gene cloned into pVR1012 vector (form A[TM-Cter])

The truncated gB gene of PRV strain NIA3 was amplified by PCR using the following primers, using the viral DNA as a template:

SB201 (SEQ ID NO: 55) and

SB203 (39 members) (SEQ ID NO: 57) ' -TTTTAAAGGATCCCTAGTGGTCCACCTTGACCACGCGGGTC-3’ .

The amplification product (2262 bp) was digested with EcoRV and BamHI enzymes and then cloned into the pVR1012 vector (Example 2) previously digested with EcoRV and BamHI, resulting in the plasmid pSB102 of approximately 7142 bp in length.

The truncated form of the gB gene (A[TM-Cter]) encodes a 750 amino acid protein containing the extracellular domain of the PRV gB glycoprotein.

8.1.3 pNS009: gB gene cloned into pABHO vector (tPA A[TM-Cter] form)

The tPA A[TM-Cter] form of the gB gene of PRV strain NIA3 was amplified by PCR from the pSB101 template (Example 8.1.1) using the following primers:

SB203 (SEQ ID NO: 57) and

SB217 (39 members) (SEQ ID NO: 58) 5'-AAAATTTCGATATCCACCTCGGCCTCGCCGACGCCCGGG-3'.

The amplification product (2088 bp) was digested with EcoRV and BglII enzymes, and then previously digested with EcoRV and BglII.

- 65 was cloned into the digested pAB110 vector (Example 2), resulting in the plasmid pNS009 of approximately 7127 bp in length.

The tPA Α[TM-Cter] form of the gB gene encodes a 720 amino acid long protein containing the extracellular domain of the PRV gB glycoprotein.

8.2 Plasmids encoding different forms of PRV-gC

8.2.1 pSB103: gC gene cloned into pVR1012 vector (native form)

The gC gene of PRV strain NIA3 was amplified by PCR using the following primers, using the viral DNA as a template:

SB204 (36 members) (SEQ ID NO: 59) 5'-TTTTAAGATATCATGGCCTCGCTCGCGCGTGCGATG-3' and SB205 (37 members) (SEQ ID NO: 60) 5'-TTTTAAAGATCTTTAAGGCCCCGCCTGGCGGGTAGTAG-3'.

The amplification product (1452 bp) was digested with EcoRV and BglII enzymes and then cloned into the pVR1012 vector (Example 2) previously digested with EcoRV and BglII, resulting in the plasmid pSB103 of approximately 6323 bp in length.

The PRV gC gene encodes a glycoprotein of 479 amino acids in length.

8.2.2 pSB104: gC gene cloned into pVR1012 vector (A[TM-Cter] form)

The truncated gC gene of PRV strain NIA3 was amplified by PCR using the following primers, using viral DNA as a template:

- 66 SB204 (sequence ID 59) and

SB203 (36 members) (SEQ ID NO: 61) 5'-TTTTAAAGACTTTAGGGGGAGGCGTCGTAGCGCTG-3'.

The amplification product (1332 bp) was digested with EcoRV and BglII enzymes and then cloned into the pVR1012 vector (Example 2) previously digested with EcoRV and BglII, resulting in the plasmid pSB104 of approximately 6206 bp in length.

The truncated form of the gC gene (A[TM-Cter]) encodes a 440 amino acid protein that contains the extracellular domain of the PRV gC glycoprotein.

8.2.3 pNS012: gC gene cloned into pABl10 vector (tPA A[TM-Cter] form)

The tPA A[TM-Cter] form of the gC gene of PRV strain NIA3 was amplified by PCR from the pSB103 template using the following primers (Example 8.2.1):

SB206 (SEQ ID NO: 61) and

SB218 (39 members) (SEQ ID NO: 62) 5'-AAAATTTCGATATCCACGGCGCTCGGCACGACGACGCCCAAC-3'.

The amplification product (1270 bp) was digested with EcoRV and BglII enzymes and then cloned into the pABHO vector (Example 2) previously digested with EcoRV and BglII, resulting in the plasmid pNS012 of approximately 6311 bp in length.

The tPA A[TM-Cter] form of the gC gene encodes a 448 amino acid long protein that contains the extracellular domain of the PRV gC glycoprotein.

8.3 Plasmids encoding different forms of PRV-gD

8.3.1 pSB105: gD gene cloned into pVR1012 vector (native form)

The gD gene of PRV strain NIA3 was amplified by PCR using the following primers, using the viral DNA as a template:

SB207 (36 members) (SEQ ID NO: 63) 5'-AATTTTGATATCATGCTGCTCGCAGCGCTATTGGCG-3' and SB208 (36 members) (SEQ ID NO: 64) 5'-AATTTTGGATCCCTACGGACCGGGCTGCGCTTTTAG-3'.

The amplification product (1227 bp) was digested with EcoRV and BamHI enzymes and then cloned into the pVR1012 vector (Example 2) previously digested with EcoRV and BamHI, resulting in the plasmid pSB105 of approximately 6104 bp in length.

The PRV gD gene encodes a glycoprotein of 404 amino acids in length.

8.3.2 pSB106: gD gene cloned into pVR1012 vector (A[TM-Cter] form)

The truncated gD gene of PRV strain NIA3 was amplified by PCR using the following primers, using the viral DNA as a template:

SB207 (SEQ ID NO: 63) and

SB209 (40 members) (SEQ ID NO: 65) 5'-AAATTTTGGATCCCTAGCGGTGGCGCGAGACGCCCGGCGC-3'.

The amplification product (1077 bp) was digested with EcoRV and BamHI enzymes and then cloned into the pVR1012 vector (Example 2) previously digested with EcoRV and BamHI.

- 68 resulting in the plasmid pSB106 of approximately 5957 bp in length.

The truncated form of the gD gene (A[TM-Cter]) encodes a 355 amino acid protein containing the extracellular domain of the PRV gD glycoprotein.

8.3.3 pPB238: gD gene cloned into pABHO vector (tPA A[TM-Cter] form)

The tPA A[TM-Cter] form of the gD gene of PRV strain NIA3 was amplified by PCR from the pSB105 template using the following primers (Example 8.3.1):

SB209 (SEQ ID NO: 65) and

SB219 (39 members) (SEQ ID NO: 66) 5'-AAAATTTCGATATCCACCTTCCCCCCGCCGCTACCCCG-3'.

The amplification product (1015 bp) was digested with EcoRV and BamHI enzymes and then cloned into the pABHO vector (Example 2) previously digested with EcoRV and BamHI, resulting in the plasmid pPB238 of approximately 6056 bp in length.

The tPA A[TM-Cter] form of the gD gene encodes a 363 amino acid long protein that contains the extracellular domain of the PRV gD glycoprotein.

Example 9

Plasmids encoding different forms of antigens from the Lelystad strain of porcine reproductive respiratory syndrome virus (PRRSV)

The genes encoding the ORF3, ORF5 and ORF6 proteins of PRRSV were prepared by RT-PCR from the Lelystad strain [J. Mulenberg et al., Virology 19, 62-72 (1993); WO-A-21375; deposited in

- 69 dated 5 June 1991, under number 1-1102, at the following institution: National Collection of Microorganism Cultures (CNCM), Pasteur Institute, Paris, France] from viral RNA.

9.1 Plasmids encoding different forms of ORF3 from the Lelystad strain of PRRSV

9.1.1 pLF1009: ORF3 gene cloned into pVR1012 vector (native form)

The cDNA of the ORF3 gene of the Lelystad strain was synthesized based on the corresponding viral RNA using primer LF028 and then amplified by PCR using the following oligonucleotide pair:

LF027 (30 members) (SEQ ID NO: 67) 5'-CACTACGATATCATGGCTCATCAGTGTGCA-3' and LF028 (30 members) (SEQ ID NO: 68) 5'-CACTACAGATCTTTATCGTGATGTACTGGG-3'.

The 802 bp DNA fragment obtained by digesting the PCR product with EcoRV and BglIII was ligated to the 4879 bp fragment obtained by digesting pVR1012 (Example 2) with EcoRV and BglIII, resulting in the approximately 5681 bp plasmid pLF1009.

The ORF3 gene from the Lelystad strain of PRRSV encodes a protein of 265 amino acids.

9.2 Plasmids encoding different forms of ORF5 from the Lelystad strain of PRRSV

9.2.1 pLFIO11; ORF5 gene cloned into pVR1012 vector (native form)

The cDNA of the ORF5 gene of the Lelystad strain was synthesized based on the corresponding viral RNA using primer LF020 and then amplified by PCR using the following oligonucleotide pair:

LF019 (30 members) (SEQ ID NO: 69) 5'-CTCACCGTCGACATGAGATGTTCTCACAAA-3' and LF020 (30 members) (SEQ ID NO: 70) 5'-CTCACCTCTAGACTAGGCCTCCCATTGCTC-3'.

The 802 bp DNA fragment obtained by digesting the PCR product with SalI and XbaI was ligated to the 4879 bp fragment obtained by digesting pVR1012 (Example 2) with SalI and XbaI, resulting in the approximately 5681 bp plasmid pLF1011.

The ORF5 gene from the Lelystad strain of PRRSV encodes a protein of 201 amino acids in length.

9.2.2 pLF1012: ORF5 gene cloned into pABHO vector (truncated form)

The ORF5 gene deleted for the transmembrane and carboxy-terminal domains was synthesized by PCR reaction from the pLF101 template (Example 9.2.1) using the following oligonucleotide pair:

LF021 (30 members) (sequence ID 71)

5'-CACCTCGGATCCTTTGCCGATGGCAACGGC-3' and

- 71 LF022 (33 members) (SEQ ID NO: 72) 5'-CACCTCGGATCCTTAGACTTCGGCTTTGCCCAA-3'.

The 432 bp DNA fragment obtained by digesting the PCR product with BamHI was ligated to the 5105 bp fragment obtained by digesting pABHO (Example 2) with BamHI, resulting in the approximately 5537 bp plasmid pLF1012.

The thus modified ORF5 gene (tPA λ[TM+Cter]) from the Lelystad strain of PRRSV encodes a protein of 168 amino acids.

9.3 Plasmids encoding different forms of ORF6 from the Lelystad strain of PRRSV

9.3.1 pLF1013: ORF6 gene cloned into pVR1012 vector (native form)

The cDNA of the ORF6 gene of the Lelystad strain was synthesized based on the corresponding viral RNA using primer LF024 and then amplified by PCR using the following oligonucleotide pair:

LF023 (30 members) (SEQ ID NO: 73) 5'-CACTCAGTCGACATGGGAGGCCTAGACGAT-3' and LF024 (30 members) (SEQ ID NO: 74) 5'-CACTCATCTAGATTACCGGCCATACTTGAC-3'.

The 528 bp DNA fragment obtained by digesting the PCR product with SalI and XbaI was ligated to the 4481 bp fragment obtained by digesting pVR1012 (Example 2) with SalI and XbaI, resulting in the approximately 5409 bp plasmid pLF1013.

- 72 The ORF6 gene from the Lelystad strain of PRRSV encodes a protein of 173 amino acids.

9.3.2 pLF1014: ORF6 gene cloned into pABHO vector (truncated form)

The ORF6 gene deleted for the transmembrane and carboxy-terminal domains was synthesized by PCR reaction from the pLF1013 template (Example 9.3.1) using the following oligonucleotide pair:

LF025 (30 members) (sequence ID 75)

5'-CACTACGGATCCGTGTCACGCGGCCGACTC-3' and

LF026 (33 members) (SEQ ID NO: 76) 5'-CACTACGGATCCTTAAACAGCTCGTTTGCCGCC-3'.

The 390 bp DNA fragment obtained by digesting the PCR product with BamHI was ligated to the 5105 bp fragment obtained by digesting pABHO (Example 2) with BamHI, resulting in the approximately 5495 bp plasmid pLF1014.

The thus modified ORF6 gene (tPA A[TM+Cter]) from the PRRSV Lelystad strain encodes a protein of 154 amino acids.

Example 10

Plasmids encoding different forms of antigens from porcine reproductive respiratory syndrome virus (PRRSV) ATCC VR-2332 American strain

The genes encoding the ORF3, ORF5 and ORF6 proteins of PRRSV were prepared by RT-PCR from the viral RNA of the American strain [M. Martaugh et al., Arch. Virol. 140, 4151-1460 (1995); deposited with the ATCC under number VR-2332].

10.1 Plasmids encoding different forms of ORF3 from PRRSV strain VR-2332

10.1.1 pLF1015: ORF3 gene cloned into pVR1012 vector (native form)

The cDNA of the ORF3 gene of strain VR-2332 was synthesized based on the corresponding viral RNA using primer LF038 and then amplified by PCR using the following oligonucleotide pair:

LF037 (30 members) (SEQ ID NO: 77) '-CACTACGATATCATGGTTAATAGCTGTACA-3' and

LF038 (30 members) (SEQ ID NO: 78) 5’-CACTACTCTAGACTATCGCCGTACGGCACT-3'.

The 769 bp DNA fragment obtained by digesting the PCR product with EcoRV and XbaI was ligated to the 4900 bp fragment obtained by digesting pVR1012 (Example 2) with EcoRV and XbaI, resulting in the approximately 5669 bp plasmid pLF1015.

The ORF3 gene from PRRSV strain VR-2332 encodes a protein of 254 amino acids.

10.2 Plasmids encoding different forms of ORF5 from PRRSV strain VR-2332

10.2.1 pLF1017: ORF5 gene cloned into pVR1012 vector (native form)

The cDNA of the ORF5 gene of the VR-2332 strain was synthesized based on the corresponding viral RNA using primer LF030 and then amplified by PCR using the following oligonucleotide pair:

- 74 LF029 (30 members) (SEQ ID NO: 79) 5'-CACTACGATATCATGTTGGAGAAATGCTTG-3' and LF030 (30 members) (SEQ ID NO: 80) 5'-CACTACAGATCTCTAAGGACGACCCCATTG-3'.

The 607 bp DNA fragment obtained by digesting the PCR product with EcoRV and BglII was ligated to the 4879 bp fragment obtained by digesting pVR1012 (Example 2) with EcoRV and BglII, resulting in the approximately 5486 bp plasmid pLF1017.

The ORF5 gene from PRRSV strain VR-2332 encodes a protein of 200 amino acids in length.

10.2.2 pLF1018: ORF5 gene cloned into pABl10 vector (truncated form)

The ORF5 gene deleted for the transmembrane and carboxy-terminal domains was synthesized by PCR reaction from the pLF1017 template (Example 10.2.1) using the following oligonucleotide pair:

LF031 (30 members) (SEQ ID NO: 81) 5'-CACTACGGATCCGCCAGCAACGACAGCAGCTCC-3' and LF032 (33 members) (SEQ ID NO: 82) 5'-CACTACGGATCCTTAGACCTCAACTTTGCCCCT-3'.

The 426 bp DNA fragment obtained by digesting the PCR product with BamHI was ligated to the 5105 bp fragment obtained by digesting pABHO (Example 2) with BamHI, resulting in the approximately 5531 bp plasmid pLF1018.

- 75 The thus modified ORF5 gene (tPA A[TM+Cter]) from PRRSV strain VR-2332 encodes a protein of 166 amino acids in length.

10.3 Plasmids encoding different forms of ORF6 from PRRSV strain VR-2332

10.3.1 pLF1019: ORF6 gene cloned into pVR1012 vector (native form)

The cDNA of the ORF6 gene of strain VR-2332 was synthesized based on the corresponding viral RNA using primer LF034 and then amplified by PCR using the following oligonucleotide pair:

LF033 (33 members) (SEQ ID NO: 83) 5'-CACATCCTGCAGATGGGGTCGTCCTTAGATGAC-3' and LF034 (30 members) (SEQ ID NO: 84) 5'-CACATCTCTAGATTATTTGGCATATTTGAC-3'.

The 527 bp DNA fragment obtained by digesting the PCR product with PstI and XbaI was ligated to the 4871 bp fragment obtained by digesting pVR1012 (Example 2) with PstI and XbaI, resulting in the approximately 5398 bp plasmid pLF1019.

The ORF6 gene from PRRSV strain VR-2332 encodes a protein of 174 amino acids.

10.3.2 pLF1016: ORF6 gene cloned into pABl10 vector (truncated form)

The ORF6 gene deleted for the transmembrane and carboxy-terminal domains was synthesized by PCR reaction in pLF1019.

- from 76 templates (example 10.3.1), using the following oligonucleotide pair:

LF035 (30 members) (SEQ ID NO: 85) 5'-CACTACGGATCCGTGAGTCGCGGCCGACTG-3' and

LF036 (33 members) (SEQ ID NO: 86) 5'-CACTACGGATCCTTAAACAGCTTTTCTGCCACC-3'.

The 390 bp DNA fragment obtained by digesting the PCR product with BamHI was ligated to the 5105 bp fragment obtained by digesting pABHO (Example 2) with BamHI, resulting in the plasmid pLF1016 of approximately 5459 bp in length.

The thus modified ORF6 gene (tPA Δ[TM+Cter]) from PRRSV strain VR-2332 encodes a protein of 154 amino acids.

Example 11

Plasmids encoding different forms of antigens from the HIN1 strain of swine influenza virus (SIV)

The genes encoding the hemagglutinin antigen (HA) and neuraminidase antigen (NA) of the swine influenza virus H1N1 were prepared by RT-PCR from the viral RNA of the “SW HINl” strain. The strains are available from the Center for Virology Research, Armand-Frappier Institute, University of Quebec (Laval, Canada) [D.S. Arora et al., Virus Genes 14, 251-254 (1997)]. See also G.W. Both et al., Proc. Natl. Acad. Sci. USA 80, 6996-7000 (1983).

11.1 Plasmids encoding different forms of HA from the HIN1 strain of SIV

11.1.1 pLF1001: HA gene cloned into pVR1012 vector (native form)

The cDNA of the HA gene of the HIN1 strain was synthesized based on the corresponding viral RNA using primer LF004 and then amplified by PCR using the following oligonucleotide pair:

LF003 (30 members) (SEQ ID NO: 87) 5'-CTCCATGATATCATGGAAGCAAAACTATTC-3' and LF004 (30 members) (SEQ ID NO: 88) 5'-CTCCATCAGATCTTAAATGCATATTCTGCA-3'.

The 1705 bp DNA fragment obtained by digesting the PCR product with EcoRV and BglII was ligated to the 4879 bp fragment obtained by digesting pVR1012 (Example 2) with EcoRV and BglII, resulting in the approximately 6584 bp plasmid pLF1001.

The HA gene from the SIV HIN1 strain encodes a protein of 566 amino acids in length.

11.1.2 pLF1002: HA gene cloned into pLF999 vector (modified form)

The HA gene deleted for the transmembrane and carboxy-terminal domains was synthesized by PCR reaction from the pLF1001 template (Example 11.1.1) using the following oligonucleotide pair:

LF005 (30 members) (sequence ID 89)

5'-TCCGCGGCCGCACATGCTAACAATTCCACA-3' and

- 78 LF006 (32 members) (SEQ ID NO: 90) 5’-TCCGCGGCCGCTTACATTGATTCTAGTTTCAC-3’.

The 1515 bp DNA fragment obtained by digesting the PCR product with NotI was ligated to the 5678 bp fragment obtained by digesting pLF999 (Example 2) with NotI, resulting in the 7193 bp plasmid pLF1002.

The thus modified HA gene (rabbit globin gene intron II, tPA, Δ[TM+Cter]) from the SIV HIN1 strain encodes a protein of 530 amino acids.

11.2 Plasmids encoding different forms of NA from the HIN1 strain of SIV

11.2.1 pLF1003: NA gene cloned into pVR1012 vector (native form)

The cDNA of the NA gene of the HIN1 strain was synthesized based on the corresponding viral RNA using primer LF008 and then amplified by PCR using the following oligonucleotide pair:

LF007 (30 members) (sequence ID 91)

5'-CACCTGGTCGACATGAATCCAAATCAGAAG-3' and

LF008 (30 members) (SEQ ID NO: 92) 5'-CACCTGTCTAGACTACTTGTCAATGGTGAA-3'.

The 1416 bp DNA fragment obtained by digesting the PCR product with SalI and XbaI was ligated to the 4881 bp fragment obtained by digesting pVR1012 (Example 2) with SalI and XbaI, resulting in the approximately 6297 bp plasmid pLF1003.

- 79 The NA gene from the HIN1 strain of SIV encodes a protein of 469 amino acids in length.

11.2.2 pLF1004: NA gene cloned into pLF999 vector (modified form)

The NA gene deleted for the transmembrane and carboxy-terminal domains was synthesized by PCR reaction from the pLF1003 template using the following oligonucleotide pair:

LF009 (31 members) (SEQ ID NO: 93) ' -CACTACGAATTCACAAATTGGGAATCAAAAT-3 ' and

LF010 (30 members) (SEQ ID NO: 94) 5'-AATTTGTGAATTCGCGGCCGGGATCCGGT-3'.

The 1207 bp DNA fragment obtained by digesting the PCR product with EcoRI was ligated to the 5678 bp fragment obtained by digesting pLF999 (Example 2) with EcoRI, resulting in the approximately 6885 bp plasmid pLF1004.

The thus modified NA gene (rabbit globin gene intron II, tPA, Δ[TM+Cter]) from the SIV HIN1 strain encodes a protein of 431 amino acids.

Example 12

Plasmids encoding different forms of antigens from the H3N2 strain of swine influenza virus (SIV)

The genes encoding the HA and NA antigens of the H3N2 swine influenza virus were prepared by RT-PCR from the viral RNA of the “Côtes du Nord 1987 (cdn87)” strain mentioned by the World Health Organization (WHO). The strain was obtained from the Virology Laboratory of the National Influenza Reference Center (8

- 80 avenue Rockefeller, 69008 Lyon. France) can be obtained.

12.1 Plasmids encoding different forms of HA from the SIV H3N2 strain

12.1.1 pLF1005: HA gene cloned into pVR1012 vector (native form)

The cDNA of the HA gene of the H3N2 strain was synthesized based on the corresponding viral RNA using primer LF012 and then amplified by PCR using the following oligonucleotide pair:

LF011 (30 members) (SEQ ID NO: 95)

5'-CTGCACGTCGACATGAAGACTGTCATTGCC-3' and

LF012 (24 members) (SEQ ID NO: 96) 5'-GATATCTCAGATGCAAATGTTGCA-3'.

The 1709 bp DNA fragment obtained by digesting the PCR product with EcoRV and SalI was ligated to the 4893 bp fragment obtained by digesting pVR1012 (Example 2) with EcoRV and SalI, resulting in the approximately 6602 bp plasmid pLF1005.

The HA gene from the SIV H3N2 strain encodes a protein of 566 amino acids in length.

12.1.2 pLF1006: HA gene cloned into pLF999 vector (modified form)

The HA gene deleted for the transmembrane and carboxy-terminal domains was synthesized by PCR reaction from the pLF1005 template (Example 12.1.1) using the following oligonucleotide pair:

- 81 LF013 (33 members) (SEQ ID NO: 97) 5'-CACCGCGGATCCCTTCCAGAAAATGGCAGCACA-3' and LF014 (33 members) (SEQ ID NO: 98) 5'-CACCGCGGATCCTTAGTCTTTGTATCCCCGACTT-3'.

The 1542 bp DNA fragment obtained by digesting the PCR product with BamHI was ligated to the 5678 bp fragment obtained by digesting pLF999 (Example 2) with BamHI, resulting in the approximately 7220 bp plasmid pLF1006.

The thus modified HA gene (rabbit globin gene intron II, tPA, A[TM+Cter]) from the SIV H3N2 strain encodes a protein of 538 amino acids.

12.2 Plasmids encoding different forms of NA from the SIV H3N2 strain

12.2.1 pLF1007: NA gene cloned into pVRl012 vector (native form)

The cDNA of the NA gene of the H3N2 strain was synthesized based on the corresponding viral RNA using primer LF016 and then amplified by PCR using the following oligonucleotide pair:

LF015 (30 members) (SEQ ID NO: 99) '-CACTCAGATATCATGAATCCAAAGCAAAAG-3' and

LF016 (30 members) (sequence ID 100) ' -CACTCATCTAGATTATATAGGCATGAGATC-3 ' .

The 1414 bp DNA fragment obtained by digesting the PCR product with EcoRV and XbaI was ligated to the 4900 bp fragment obtained by digesting pVR1012 (Example 2) with EcoRV and XbaI, resulting in

- 82 times, the plasmid pLF1007, which is approximately 6314 bp in length, was obtained.

The NA gene from the SIV H3N2 strain encodes a protein of 469 amino acids.

.2.2 pLF1008: NA gene cloned into pLF999 vector (modified form)

The NA gene deleted for the transmembrane and carboxy-terminal domains was synthesized by PCR reaction from the pLF1007 template (Example 12.2.1) using the following oligonucleotide pair:

LF017 (33 members) (SEQ ID NO: 101)

5'-CACTACGGATCCTTCAAGCAATATGAGTGCGAC-3' and

LF018 (33 members) (SEQ ID NO: 102) ' -CACTACGGATCCTTATGAAGTCCACCATACTCT-3 ' .

The 1221 bp DNA fragment obtained by digesting the PCR product with BamHI was ligated to the 5678 bp fragment obtained by digesting pLF999 (Example 2) with BamHI, resulting in the approximately 6899 bp plasmid pLF1008.

The thus modified NA gene (rabbit globin gene intron II, tPA, Á[TM+Cter]) from the SIV H3N2 strain encodes a protein of 431 amino acids.

Example 13

Plasmid encoding bovine GM-CSF

The cDNA of the bovine GM-CSF gene was synthesized from cellular RNA from bovine mononuclear blood cells using primer LF065 and then amplified as follows:

- amplified by PCR reaction using 83 bi oligonucleotide pairs:

LF054 (36 members) (SEQ ID NO: 103)

5'-CATATCGTCGACATGTGGCTGCAGAACCTGCTTCTC-3' and

LF055 (34 members) (SEQ ID NO: 104) 5’-CATGACCAGATCTTCACTTCTGGGCTGGTTCCCA-3’.

The 437 bp DNA fragment obtained by digesting the PCR product with SalI and BglII was ligated to the 4860 bp fragment obtained by digesting pVR1012 (Example 2) with SalI and BglII, resulting in plasmid pLF1032 (approximately 5297 bp in length).

The bovine GM-CSF gene encodes a protein of 143 amino acids in length.

Example 14

Plasmid encoding porcine GM-CSF

The cDNA of the porcine GM-CSF gene was synthesized from cellular RNA from porcine mononuclear blood cells using primer LF067 and then amplified by PCR using the following oligonucleotide pair:

LF056 (36 members) (SEQ ID NO: 105)

5'-CATATCGTCGACATGTGGCTGCAGAACCTGCTTCTC-3' and

LF057 (37 members) (SEQ ID NO: 106) 5'-CATGACCAGATCTTCACTTCTGGGCTGGTTCCCAGCA-3' .

The 440 bp DNA fragment obtained by digesting the PCR product with SalI and BglII was ligated to the 4860 bp fragment obtained by digesting pVR1012 (Example 2) with SalI and BglII, resulting in

- 84 times, the plasmid pLF1033 (approximately 5300 bp long) was obtained.

The porcine GM-CSF gene encodes a protein of 144 amino acids in length.

Example 15

Formulation of vaccine plasmids

DNA solutions containing one or more plazinides according to Examples 13 and 14 were concentrated by ethanol precipitation (Id. Sambrook et al., 1989). The DNA pellet was taken up in 0.9% NaCl solution to obtain a 1 mg/ml solution. The 0.75 mM DMRIE-DOPE solution was prepared by taking up the DMRIE-DOPE lyophilisate in an appropriate volume of sterile H ₂ 0.

To form plasmid DNA-lipid complexes, a 0.75 mM DMRIE-DOPE solution was diluted in equal parts with a 1 mg/ml DNA solution in 0.9 mM NaCl. The DNA solution was gradually added along the wall of the vial containing the cationic lipid solution using a 26G needle to avoid foaming. Once the two solutions were mixed, they were gently shaken together. Finally, a preparation of 0.375 mM DMRIE-DOPE and 500 μg/ml plasmid was obtained.

It is desirable to keep the solutions at room temperature during the above operations. In order to allow the formation of the DNA/DMRIE-DOPE complex, the mixture is allowed to stand at room temperature for 30 minutes before immunizing the animals.

Example 16

Cattle vaccinated against BHV-1 were randomly divided into 3 groups.

Group 1 is the control group.

In group 2, animals are administered a mixture containing the vaccine plasmids pPB281 (BHV-1 gB, A[TmCter] form, example 3.1.2), pPB292 (BHV-1 gC, A[Tm-Cter] form, example 3.2.2) and pPB284 (BHV-1 gD, A[Tm-Cter] form, example 3.3.2).

In group 3, the animals were given the mixture used in group 2, but formulated with DMRIE-DOPE as described in example 15.

For cattle vaccination, a 10 ml injection is used, which is administered intramuscularly using syringes with needles, and the vaccination is repeated after 21 days. The total amount of plasmid used during each immunization is 1500 μg per plasmid.

A person skilled in the art has the appropriate expertise to adjust the volume or concentration to the required plasmid dose.

Serological responses induced by two vaccine plasmid mixtures expressing the BHV-1 gB, gC and gD antigens are monitored for 35 days after the first vaccination.

- 86 The results are shown in the table below:

Plasmids Formulation Antigens Dose SN is the 28th. SN is 35. day day control — — — 0.2 ± 0.0 0.2 ± 0.0 pPB281 — gB A[Tm-Cter] 1500 μg 1.0 ± 0.5 1.2 ± 0.8 pPB292 gC Á[Tm-Cter] 1500 μg pPB294 gD Á[Tm-Cter] 1500 μg pPB281 DMRIE-DOPE gB A[Tm-Cter] 1500 μg 2.1 ± 0.6 2.7 ± 0.6 pPB292 gC Á[Tm-Cter] 1500 μg pPB294 gD A[Tm-Cter] 1500 μg

Example 17

Immunization of pigs against PRV

15 pigs, approximately 7 weeks old, were randomly divided into 3 groups.

Group 1 is the control group.

In group 2, animals are administered a mixture containing the vaccine plasmids pNS009 (PRV gB, tPA A[Tm-Cter] form, example 8.1.3), pNS012 (PRV gC, tPA A[Tm-Cter] form, example 8.2.3) and pPB238 (PRV gD, tPA A[Tm-Cter] form, example 8.3.3).

In group 4, the animals were given the mixture used in group 3, but formulated with DMRIE-DOPE as described in Example 15, such that the final concentration of DMRIE-DOPE was 0.0535 mM.

- 87 The 350-350 μg plasmid required for each vaccination protocol is mixed in a final volume of 14 ml.

To vaccinate pigs, a 2 ml injection is used, which is administered intramuscularly using syringes with a needle, and the vaccination is then repeated after 21 days.

On day 35, pigs were given 2 ml intranasally - 1 ml per nostril - of PRV NIA3 virus strain (titer: 10 ^{7 '76} CCID ₅₀ /ml).

The body weight of the animals is measured for 42 days after the first vaccination.

The relative body weight gain (G7) of the animals is calculated for 7 days after PRV virus administration by taking the difference between the body weight measured on day 7 (D7) and the body weight measured on the day of PRV virus administration (D0), then dividing it by the body weight measured on the day of PRV virus administration and expressing it as a daily percentage:

(body weight on day 7 - body weight on day 0) * 100 / (body weight on day 0 * 7)

AG7 is the difference between the relative weight gain averages of vaccinated and control animals.

- 88 The results are shown in the table below:

Plasmids Wording Antigens Dose Average weight on day 35 Average weight of ΔΘ7 42. day control — — — 25.3 ± 6.2 22.0 ±5.0 — pNS009 — gB A[TM-Cter]tPA 350 μg 25.3 ± 4.8 26.1 ± 4.7 2.46 pNS0012 gC A[TM-Cter]tPA 350 μς pPB238 gD ΔίΤΜ-Cter]tPA 350 μg pNS009 DMRIE-DOPE gB ΔΙΤΜ-Cter]tPA 350 μg 23.8 ± 4.5 26.2 ±4.9 3.41 pNS0012 gC ΔΙΤΜ-Cter]tPA 350 μς pPB238 gD ΔΙΤΜ-Cter]tPA 350 μg

It is obvious that the scope of the invention defined in the patent claims accompanying the description is not limited to the specific embodiments described in the description, but includes all those variations that do not depart from the inventive concept and the scope of the invention.

Claims (40)

PATENT CLAIMS 1. A DNA vaccine against a pathogen infecting domestic animals, in particular cattle and pigs, comprising a plasmid containing a nucleotide sequence encoding an immunogen of a pathogen of the given animal species in a manner that allows the in vivo expression of the sequence, and further comprising a cationic lipid comprising a quaternary ammonium salt of the following general formula I: CH ₃ Ri-O-CH ₂ -CH-CH ₂ -N ⁺ -R ₂ -X (I) ORi CH ₃ where in the formula R1 is a saturated or unsaturated, straight-chain aliphatic group of 12-18 carbon atoms, _R2 is another aliphatic group of 2-3 carbon atoms, and X is a hydroxyl or amino group, and this lipid is preferably DMRIE. 2. The vaccine according to claim 1, characterized in that it also contains DOPE. 3. The vaccine according to claim 1 or 2, characterized in that it also contains GM-CSF of the given animal species. 4. The vaccine according to claim 1 or 2, characterized in that it also contains an expression vector containing the gene encoding the GM-CSF of the given animal species in such a way that - 90 which allows for the in vivo expression of this sequence. 5. The vaccine according to claim 4, characterized in that the expression vector is a plasmid. 6. The vaccine according to any one of claims 1-5, characterized in that the nucleotide sequence encoding the immunogen of the pathogen is a sequence derived from a gene from which the part encoding the transmembrane domain has been deleted. 7. Vaccine according to any one of claims 1-6, characterized in that the plasmid containing the nucleotide sequence encoding the immunogen of the pathogen also contains a heterologous signal sequence, preferably a nucleotide sequence encoding tPA. 8. The vaccine according to any one of claims 1-7, characterized in that the plasmid containing the nucleotide sequence encoding the immunogen of the pathogen also contains a stabilizing intron. 9. The vaccine according to claim 7, wherein the intron is intron II of the rabbit beta-globin gene. 10. The vaccine according to any one of claims 1-5, characterized in that it contains a BHV-1 nucleotide sequence. 11. The vaccine according to claim 10, characterized in that it contains the sequence of the gB gene, which has been optimized in such a way that the sequence of the signal peptide of the gB glycoprotein has been replaced by a signal sequence, preferably the human tPA signal sequence, and/or the DNA fragment encoding the gB transmembrane domain has been deleted. 12. The vaccine according to claim 10, characterized in that it contains the sequence of the gC gene, which has been optimized in such a way that the sequence of the signal peptide of the gC glycoprotein has been combined with a signal sequence, preferably the human tPA signal sequence, and/or the DNA fragment encoding the gC transmembrane domain has been deleted from it. 13. The vaccine according to claim 10, characterized in that it contains the sequence of the gD gene, which has been optimized in such a way that the sequence of the signal peptide of the gD glycoprotein has been replaced by a signal sequence, preferably the human tPA signal sequence, and/or the DNA fragment encoding the gD transmembrane domain has been deleted. 14. The vaccine of claim 10, characterized in that it comprises DMRIE-DOPÉ, an expression plasmid encoding the BHV-1 gB antigen, which has been optimized by deleting the nucleotide sequence fragment encoding the transmembrane domain and the adjacent C-terminal part thereof; another expression plasmid encoding the BHV-1 gC antigen, which has been optimized by deleting the nucleotide sequence fragment encoding the transmembrane domain and the adjacent C-terminal part thereof; and a third expression plasmid encoding the BHV-1 gD antigen, which has been optimized by deleting the nucleotide sequence fragment encoding the transmembrane domain and the adjacent C-terminal part thereof. 15. The vaccine according to any one of claims 1-5, characterized in that it comprises a BRSV nucleotide sequence. 16. The vaccine according to claim 15, characterized in that it contains the sequence of the F gene of BRSV, which has been optimized in such a way that the signal sequence of the BRSV F-white protein has been replaced by a signal sequence, preferably the signal sequence of human tPA, and/or the DNA fragment encoding the F transmembrane domain has been deleted. 17. The vaccine according to claim 15, characterized in that it contains the sequence of the G gene of BRSV, which has been optimized in such a way that the signal sequence of the G glycoprotein of BRSV has been replaced by a signal sequence, preferably the signal sequence of human tPA, and/or the DNA fragment encoding the transmembrane domain of G has been deleted. 18. The vaccine according to claim 15, characterized in that it comprises DMRIE-DOPÉ, an expression plasmid encoding the BRSV F antigen, which has been optimized by inserting the human tPA signal sequence in place of the F signal sequence and deleting the nucleotide sequence fragment encoding the F transmembrane domain and the adjacent C-terminal part thereof; and another expression plasmid encoding the BRSV Gantigen, which has been optimized by inserting the human tPA signal sequence in place of the G signal sequence and deleting the G trans - 93 nucleotide sequence fragment encoding the membrane domain and its adjacent C-terminal part. 19. The vaccine according to any one of claims 1-5, characterized in that it comprises a BVDV nucleotide sequence. 20. The vaccine according to claim 19, characterized in that it comprises the sequence of the EO gene of BVDV, which has been optimized by adding a signal sequence, preferably the signal sequence of human tPA, 5' from the nucleotide sequence encoding the EO protein, and/or by inserting an intron, preferably intron II of the rabbit beta-globin gene, 5' from the nucleotide sequence encoding the EO protein. 21. The vaccine according to claim 19, characterized in that it comprises the sequence of the E2 gene, which has been optimized in such a way that a signal sequence, preferably the signal sequence of human tPA, has been added 5' from the nucleotide sequence encoding the E2 protein, and/or the DSN fragment encoding the transmembrane domain of the E2 protein has been deleted, and/or an intron, preferably intron II of the rabbit beta-globin gene, has been inserted 5' from the nucleotide sequence encoding the E2 protein. 22. The vaccine of claim 19, characterized in that it comprises DMRIE-DOPÉ, an expression plasmid encoding the BVDV EO antigen optimized to have the human tPA signal sequence inserted 5' from the EO and the rabbit beta-globin gene intron II inserted 5' from the EO; and another expression plasmid encoding the BVDV E2 - It encodes the antigen 94, which was optimized by inserting the human tPA signal sequence 5' from E2, deleting the nucleotide sequence fragment encoding the E2 transmembrane domain, and inserting intron II of the rabbit beta-globin gene 5' from E2. 23. The vaccine according to any one of claims 1-5, characterized in that it comprises a bPI-3 nucleotide sequence. 24. The vaccine according to claim 23, characterized in that it contains the sequence of the HN gene of bPI-3, which has been optimized in such a way that the signal sequence of HN has been replaced by a signal sequence, preferably the signal sequence of human tPA, and/or the DNA fragment encoding the transmembrane domain of HN has been deleted from it, and/or an intron, preferably intron II of the rabbit beta-globin gene, has been inserted 5' from the nucleotide sequence encoding HN. 25. The vaccine according to claim 23, characterized in that it contains the sequence of the F gene of bPI-3, which is optimized in such a way that the signal sequence of F has been replaced by a signal sequence, preferably the signal sequence of human tPA, and/or the DNA fragment encoding the transmembrane domain of F has been deleted, and/or an intron, preferably intron II of the rabbit beta-globin gene, has been inserted 5' from the nucleotide sequence encoding F. 26. The vaccine according to claim 23, characterized in that it comprises DMRIE-DOPÉ, an expression plasmid encoding the HN antigen of bPI-3, which has been optimized in such a way that the human tPA signal sequence has been inserted in place of the HN signal sequence, the nucleotide sequence fragment encoding the HN transmembrane domain and the adjacent C-terminal part has been deleted, and intron II of the rabbit betaglobin gene has been inserted 5' from the HN; and another expression plasmid encoding the F antigen of bPI-3, which was optimized by inserting the human tPA signal sequence in place of the F signal sequence, deleting the nucleotide sequence fragment encoding the F transmembrane domain and the adjacent C-terminal part, and inserting intron II of the rabbit betaglobin gene 5' from F. 27. The vaccine according to any one of claims 1-5, characterized in that it comprises a PRV nucleotide sequence. 28. The vaccine according to claim 27, characterized in that it comprises the sequence of the gB gene, which has been optimized in such a way that the sequence of the signal peptide of the gB glycoprotein has been replaced by a signal sequence, preferably the human tPA signal sequence, and/or the DNA fragment encoding the gB transmembrane domain has been deleted. 29. The vaccine according to claim 27, characterized in that it comprises the sequence of the gC gene, which is optimized so as to contain the signal peptide of the gC glycoprotein. - 96 of its sequence has been replaced by a signal sequence, preferably the human tPA signal sequence, and/or the DNA fragment encoding the gC transmembrane domain has been deleted. 30. The vaccine according to claim 27, characterized in that it comprises the sequence of the gD gene, which has been optimized in such a way that the sequence of the signal peptide of the gD glycoprotein has been replaced by a signal sequence, preferably the human tPA signal sequence, and/or the DNA fragment encoding the gD transmembrane domain has been deleted. 31. The vaccine of claim 27, characterized in that it comprises DMRIE-DOPÉ, an expression plasmid encoding the PRV gB antigen, which has been optimized by deleting the nucleotide sequence fragment encoding the transmembrane domain and the adjacent C-terminal part thereof, another expression plasmid encoding the PRV gC antigen, which has been optimized by deleting the nucleotide sequence fragment encoding the transmembrane domain and the adjacent C-terminal part thereof; and a third expression plasmid encoding the PRV gD antigen, which has been optimized by deleting the nucleotide sequence fragment encoding the transmembrane domain and the adjacent C-terminal part thereof. 32. The vaccine according to any one of claims 1-5, characterized in that it comprises a PRRSV nucleotide sequence. 33. The vaccine according to claim 32, characterized in that it comprises the sequence of the ORF3 gene, which has been optimized in such a way that the sequence of the signal peptide of the protein encoded by ORF3 has been replaced by a signal sequence, preferably the human tPA signal sequence, and/or the DNA fragment encoding the ORF3 transmembrane domain has been deleted. 34. The vaccine according to claim 32, characterized in that it comprises the sequence of the ORF5 gene, which has been optimized in such a way that the sequence of the signal peptide of the protein encoded by ORF5 has been replaced by a signal sequence, preferably the human tPA signal sequence, and/or the DNA fragment encoding the ORF5 transmembrane domain has been deleted. 35. The vaccine according to claim 32, characterized in that it comprises the sequence of the ORF6 gene, which has been optimized in such a way that the sequence of the signal peptide of the protein encoded by ORF6 has been replaced by a signal sequence, preferably the human tPA signal sequence, and/or the DNA fragment encoding the ORF6 transmembrane domain has been deleted. 36. The vaccine according to claim 27, characterized in that it comprises DMRIE-DOPÉ, an expression plasmid encoding the ORF3 antigen of PRRSV, another expression plasmid encoding the ORF5 antigen of PRRSV, which has been optimized in such a way that the ORF5 signal sequence has been replaced by the sequence of the signal peptide of human tPA and the transmembrane region has been deleted. - a nucleotide sequence fragment encoding the 98 domain and its adjacent C-terminal part; and a third expression plasmid encoding the ORF6 antigen of PRRSV, which has been optimized by replacing the ORF6 signal sequence with the sequence of the signal peptide of human tPA and deleting the nucleotide sequence fragment encoding the transmembrane domain and its adjacent C-terminal part. 37. The vaccine according to any one of claims 1-5, characterized in that it comprises an SIV nucleotide sequence. 38. The vaccine according to claim 37, characterized in that it comprises a nucleotide sequence of the HA gene, which has been optimized in such a way that the signal sequence of the HA has been replaced by a signal sequence, preferably the signal sequence of human tPA, and/or the DNA fragment encoding the transmembrane domain of the HA has been deleted, and/or an intron, preferably intron II of the rabbit beta-globin gene, has been inserted 5' from the nucleotide sequence encoding the HA. 39. The vaccine according to claim 37, characterized in that it comprises a nucleotide sequence of the NA gene, which has been optimized in such a way that the signal sequence of NA has been replaced by a signal sequence, preferably the signal sequence of human tPA, and/or the DNA fragment encoding the transmembrane domain of NA has been deleted, and/or an intron, preferably intron II of the rabbit beta-globin gene, has been inserted 5' from the nucleotide sequence encoding NA. 40. The vaccine according to claim 37, characterized in that it comprises DMRIE-DOPÉ, an expression plasmid encoding the HA antigen of SIV, which has been optimized such that the human tPA signal sequence has been inserted in place of the HA signal sequence, the nucleotide sequence fragment encoding the HA transmembrane domain and the adjacent C-terminal part has been deleted, and intron II of the rabbit beta-globin gene has been inserted 5' from the HA; and another expression plasmid encoding the SIV NA antigen, which was optimized by inserting the human tPA signal sequence in place of the NA signal sequence, deleting the nucleotide sequence fragment encoding the NA transmembrane domain and its adjacent C-terminal part, and inserting intron II of the rabbit beta-globin gene 5' from the NA. The authorized person: DANUBE Patent and Trademark Office Ltd. Dr. Ádám Svingor, patent attorney